CN101663046B - Through modifying the polypeptides of FGF 21 and its purposes - Google Patents

Through modifying the polypeptides of FGF 21 and its purposes Download PDF

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CN101663046B
CN101663046B CN200880009653.9A CN200880009653A CN101663046B CN 101663046 B CN101663046 B CN 101663046B CN 200880009653 A CN200880009653 A CN 200880009653A CN 101663046 B CN101663046 B CN 101663046B
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fgf
seq
polypeptides
mankind
amino acid
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CN101663046A (en
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汤姆士·P.·库杰克
罗伯托·马利安
安娜玛莉亚·A.·海斯·蒲楠
威廉·M.·基夫
尼克·克努森
何莉莲
杰森·品克史塔夫
瓦迪姆·克赖诺夫
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Ambrx Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]

Abstract

The present invention is provided through modifying the polypeptides of FGF 21 and its purposes.

Description

Modified FGF-21 polypeptides and its purposes
The cross reference of related application
Present application advocates U.S. provisional patent application cases the 60/921,297th and 2007 filed in 30 days March in 2007 The priority and right of U.S. provisional patent application cases the 60/988th, 060 filed on November 14, the application case is said Bright book and disclosure are incorporated herein in full.
Technical field
The present invention relates to the FGF-21 polypeptides optionally modified through at least one non-naturally encoded amino acids.
Background technology
Fibroblast growth factor is wide expression in development and mature tissue big polypeptide (Baird et al., Cancer Cells, 3：239-243,1991), and including angiogenesis, mitosis generation, pattern formation, cell point Played a crucial role in a variety of physiologic functions of change, Metabolism regulation and tissue damage reparation (McKeehan et al., Prog.Nucleic Acid Res.Mol.Biol.59：135-176,1998；Burgess, W.H. et al., Annu.Rev.Biochem.58：575-606(1989)).Prototype fibroblast growth factor (FGF) FGF-1 and FGF-2 is most It is just to be separated from the brain and hypophysis as fibroblast mitogen.FGF-3 is by mouse mammary tumor through differentiating Conventional target (Dickson et al., the Ann.N.Y.Acad.Sci.638 of viral activation：18-26(1991))；FGF-4 to FGF-6 It is oncogene products (Yoshida et al., Ann.NY Acad.Sci.638 through differentiating：27-37(1991)；Goldfarb etc. People, Ann.NYAcad.Sci 638：38-52(1991)；Coulier et al., Ann.NYAcad.Sci.638：53-61 (1991)).FGF-10 be differentiated by the polymerase chain reaction (PCR) based on homology from induced lung (Yamasaki et al., J.Biol.Chem.271：15918-15921(1996)).FGF-11 to FGF-14 (the homologous factors of FGF (FHF) 1 to 4) is to pass through Random cdna sequencing, the combination of database search and the PCR based on homology differentiate (Smallwood etc. from human retina People, Proc.Natl.Acad.Sci.USA 93：9850-9857(1996)).FGF-15 is chimera homeodomain through differentiating Downstream target (McWhirter et al., the Development 124 of cancer protein：3221-3232(1997)).FGF-16、FGF-17 Be respectively with FGF-18 differentiated by the PCR based on homology from rat heart and embryo (Miyake et al., Biochem.Biophys.Res.Commun.243：148-152(1998)；Hoshikawa et al., Biochem.Biophys.Res.Commun.244：187-191(1998)；Ohbayashi et al., J.Biol.Chem.273： 18161-18164(1998)).FGF-19 be differentiated by database search from human foetus' brain (Nishimura et al., Biochim.Biophys.Acta 1444：148-151(1999)).It has the pact that amino acid identity is about 30% to 60% The core of 120 conservative amino acid residues.
Animal model, overexpression and the analysis of naturally occurring mutation involve fibroblast growth factor and its acceptor (such as Wilkie et al., Current Biology, (1995) 5 in a variety of diseases：500-507；Pugh-Humphreys etc. People, The Cytokine Handbook, A.Thomson volumes, second edition, Academic Press, Harcourt Brace ＆ Co. publishing house, London, the 525-566 pages), show that Active Regulation can be used for treating.For example, compound suramin (Suramin) inhibitory action to FGF-2m can prevent neovascularization and the tumour growth of mouse (Pesenti et al., British Journal of Cancer, 66：367-372).Fibroblast growth factor is given birth in blood vessel Into (Lyons, M.K., et al., Brain Res. (1991) 558：315-320), wound healing (Uhl, E., et al., Br.J.Surg.(1993)80：977-980,1993), proliferation of astrocytes (astrogliosis), glial cells hyperplasia With differentiation (Biagini, G. et al., Neurochem.Int. (1994) 25：17-24), cerebral vasodilation (Tanaka, R. et al., Stroke(1995)26：Worked 2154-2159) and also during neurotrophy/nerve modulation.
Fibroblast growth factor also has a variety of positive roles, including improves blood flow and prevent calcium toxicity from improving brain Consequence (Mattson, M.P. et al., Semin.Neurosci. (1993) 5 of ischemic：295-307；Doetrocj.W.D. et al., J.Neurotrauma(1996)13：309-316).Basic FGF treatments promote the new vascular generation in ischemic myocardium (Schumacher et al., Circulation (1998) 97：645-650).Basic FGF strengthens functional rehabilitation, and promotes focal Neuron germinating (Kawamata et al., Proc.Natl.Acad.Sci. (1997) 94 (15) after cerebral infarction：8179-84).Root According to open source literature, FGF families are by least 22 member composition (Reuss et al., Cell TissueRes.313：139-157 (2003))。
Reported FGF2 1 (FGF-21) be preferentially expressed in liver (Nishimura et al., Biochimica et Biophysica Acta, 1492：203-206(2000)；WO 01/36640；With WO 01/18172, its Be incorporated herein by reference), and describe it as ischemic angiopathy, wound healing and with lung, bronchus or The treatment method of alveolar cell or the related disease of loss function and numerous other illnesss.FGF-21 be mainly expressed in liver, (referring to the U.S. Patent Publication case example 2 of No. 20040259780, the patent is quoted in full in kidney and musculature Mode be incorporated herein).FGF-21 genes are made up of 3 extrons and on chromosome 19.It is different from other FGF, FGF-21 is without propagation and oncogenic function (Genome Biol.2001；2(3)：REVIEWS3005).
U.S. Patent Publication case the 20010012628th (it is incorporated herein in entirety by reference) describes the mankind FGF-21 nucleotides and protein sequence is (respectively referring to the SEQ IDNO of U.S. Patent Publication case the 20010012628th：1 With SEQ ID NO：2).SEQ ID NO in case disclosed above：2 (being referred to as sbgFGF-19) were 209 amino acid longs, and N-terminal contains the targeting sequencing of 28 amino acid.Presented herein is SEQ ID NO：3 mankind's FGF-21 sequences are and the U.S. The SEQ ID NO of patent publication the 20010012628th：2 identical sequences.This sequence has the dried meat on position 174 The SNP (single nucleotidepolymorphism, SNP) of propylhomoserin (P), hereinafter referred to as " FGF-21 209 amino acid p-shaped formulas ".
U.S. Patent No. 6,716,626 (it is incorporated herein in entirety by reference) discusses mankind FGF-21 and its Homologous protein in its mammal (especially mouse and rat).With the U.S. Patent No. SEQ ID NO of No. 6,716,626：1 table The mouse FGF shown is highly expressed in liver and is expressed in testis and thymus gland, and shows that mankind FGF-21 may be in liver Worked in the ongoing disease and recovery of disease and/or the function of testicular function or the cell from thymus gland.U.S. Patent No. 6, The SEQ ID NO of No. 716,626：4 be 209 amino acid longs, and contains the targeting sequencing of 28 amino acid in N-terminal.This SEQ ID NO are rendered as in text：6 mankind's FGF-21 sequences are the SEQ ID NO with U.S. Patent No. 6,716,626：4 phases Same sequence.This sequence has the SNP (SNP) of the leucine (L) on position 174, hereinafter referred to as " FGF-21 209 amino acid L-shaped formulas ".
U.S. Patent Publication case the 20040259780th (it is incorporated herein in entirety by reference) discusses the mankind FGF-21, and 208 amino acid long (SEQ ID NO of U.S. Patent Publication case the 20040259780th are presented：2) and Contain the sequence of the targeting sequencing of 27 amino acid in N-terminal.Presented herein is SEQ ID NO：7 mankind's FGF-21 sequences It is the SEQ ID NO with U.S. Patent Publication case the 20040259780th：2 identical sequences.This sequence has on position The SNP (SNP) of 173 leucine (L), hereinafter referred to as " FGF-21 208 amino acid L-shaped formulas ".
Shown that FGF-21 exists in insulin stimulates the glucose in mouse 3T3-L1 fat cells to take the photograph with the absence of Take, and with dosage-dependent manner reduce ob/ob and db/db mouse and 8 week old ZDF rats after the meal with fasting blood-glucose, Triglycerides and Plasma Glucagon Level, therefore, to be provided using FGF-21 as the therapy for the treatment of diabetes and obesity Basis (WO 03/011213 being incorporated herein by reference, and Kharitonenkov et al.,JClin Invest.In June, 2005；115(6)：1627-35).Kharitonenkov et al.,J Clin Invest.In June, 2005； 115(6)：1627-35 displays that expression mankind FGF-21 transgenic mice hypoglycemia, to insulin sensitivity and resists diet The obesity of induction.Kharitonenkov et al., Endocrinology (waiting to publish) display that FGF-21 reduction diabetes Mi Glucose, triglycerides, insulin and the hyperglycemic factor of monkey (Rhesus monkey).
In addition, shown FGF-21 can effectively reduce critical patient the death rate and the incidence of disease (WO 03/059270, its with The mode of reference is incorporated herein).In U.S. patent application case 20050176631 (it is incorporated herein by reference) FGF-21 is described, it influences overall metabolic situation, and can resist in body to septicemia and by non-infectious causes for pathological The passive side effect being likely to occur during the stress reaction of caused systemic inflammatory response syndrome (SIRS).Therefore, it can be used FGF-21 reduces the death rate occurred in critical patient and the incidence of disease.Critical patient includes needing continuous, collaboration doctor, seen The unstable patient of physiology of shield and respiratory care.This kind of nursing is especially needed to be noted providing lasting monitoring and therapy drop in detail It is fixed.Critical patient includes the patient in physiological compensation imbalance risk, and therefore needs lasting monitoring to cause intensive care Team can provide immediate intervention to prevent rough sledding.Critical patient especially needs must be by that can provide Continuous Titration nursing The monitoring and life that team provides are supported.
Pegylation FGF-21 polypeptides are described in the WO 2005/091944 being incorporated herein by reference.WO FGF-21 polypeptides described in 2005/091944 are the polypeptides of 181 amino acid.With WO 2005/091944 SEQ ID NO： 1 maturation represented, wild type or natural mankind FGF-21 sequences lack targeting sequencing.This mankind FGF-21 and mouse FGF-21 (about 79% amino acid identity) and rat FGF-21 (about 80% amino acid identity) are highly consistent.Presented herein is SEQ ID NO：5 mankind's FGF-21 sequences are the SEQ IDNO with WO 05/091944：1 identical sequence.This sequence, which has, closes In the SNP (SNP) of the leucine (L) of position 146.Those skilled in the art can be easily using replacement Property mammal FGF-21 peptide sequences or the like, mutain have spreading out for enough homologys with mankind's FGF-21 sequences Biology reaches purposes as described herein.
Presented herein is SEQ ID NO：1 mankind FGF-21 sequences have the list of the proline (P) on position 146 Nucleotide polymorphisms (SNP).SEQ ID NO：1 N-terminal His labels pattern is shown as SEQ IDNO herein：2.
WO 2005/091944 describes the covalent of one or more PEG molecules and the specific residue of FGF-21 compounds Connection.Compared with natural FGF-21, gained compound be eliminate Increased Plasma Half-life and clearance rate reduction have bioactivity Pegylation FGF-21 compounds.PEG molecules are and cysteine or lysine residue are covalently attached.In cysteine Replaced to allow connection to a few PEG molecule at multiple positions.Describe one or more lysine residues (56, 59th, 69 and 122) the Pegylation at place.
Pegylation FGF-21 compounds are applicable to individual of the treatment with including but not limited to following illness：2 Patients with type Ⅰ DM, obesity, insulin resistance, hyperinsulinemia, poor glucose tolerance, hyperglycemia and metabolic syndrome. Effect can be increased compared with long circulating half-life period and by the Pegylation FGF-21ization of the less dosage of needs by reaching by taking Compound is by be especially advantageous, and it increases the possibility for needing the individual convenience of this therapy to comply with medication requirements with individual. Metabolic syndrome may be defined as at least three kinds in following sign of cluster：Abdominal obesity, in most of males, waistline is 40 English It is very little or bigger；It is at least 110 milligrams/deciliter (mg/dL) after hyperglycaemia, empty stomach；It is at least 150mg/ in high triglyceride, blood flow dL；Low HDL, less than 40mg/dL；And blood pressure is 130/85 or higher.
Be covalently attached with hydrophilic polymer polyethylene glycol (being abbreviated as PEG) is to many bioactive molecules (including egg White matter, peptide and especially hydrophobic molecule) increase is water-soluble, biological usability, increases serum half-life, increase treatment half-life period, Adjust immunogenicity, regulation bioactivity or the method for extending circulation time.PEG is widely used in medicine, artificial implantation, with And biocompatibility, non-toxic and non-immunogenicity have in the other application of importance.The property for needed for maximizing PEG, The total molecular weight and hydration status of the one or more kinds of PEG polymer being connected on bioactive molecule must it is sufficiently high with Favorable characteristics (such as increased water-soluble and circulating half-life) generally related to the connection of PEG polymer are assigned, without not The bioactivity of parent molecule is influenceed sharply.
PEG derivatives are generally for example, by lysine, cysteine and histidine residues, N-terminal and carbohydrate Part isoreactivity chemical functional group is connected with bioactive molecule.Existing grinding on the therapeutic FGF-21 compounds of formation Study carefully, but there is problem because of many reasons, one reason for this is that because protein and other molecules generally have finite population Can be used for polymer connect reactive site.Generally, it is most suitable for the site modified by polymer connection in acceptor knot Played an important role in conjunction, and be required for the bioactivity of molecule is kept.Therefore, polymer chain and bioactivity The mixed and disorderly connection of these reactive sites on molecule typically result in polymer-modified molecule bioactivity significantly reduce or Even completely lose.R.Clark et al., (1996),J.Biol.Chem., 271：21969-21977.Gather enough to be formed to have Adduct molecule amount to assign targeting molecule with the binding element of required advantage, prior art approach be usually directed to multiple polymeric arms with The random connection of molecule, so as to increase the bioactivity reduction of parent molecule or the risk even completely lost.
The reactive site for forming the tie point of PEG derivatives and protein is indicated by the structure of protein.Protein (bag Include enzyme) by multiple alpha amino acid Sequence compositions, the sequence has general structure H2N-CHR-COOH.The α amino of one amino acid Partly (H2N-) it is connected with the carboxy moiety (- COOH) of adjacent amino acid and forms that acid amides is bonded, it is represented by-(NH-CHR- CO)n-, wherein subscript " n " can be equal to hundreds of or thousands of.The fragment represented by R, which can contain, to be related to proteins biological activity and is used for Connect the reactive site of PEG derivatives.
For example, in the case of amino acid lysine, at ε and α presence-NH2Part.ε-NH2In alkaline pH Do not reacted under the conditions of value.It is to be used to connect in protein for exploitation that many technologies in protein derived field are carried out with PEG ε-the NH of the lysine residue of presence2Partial PEG derivatives.″Polyethylene Glycol andDerivatives For Advanced PEGylation ", Nektar Molecular Engineering Catalog, 2003, the 1-17 pages. However, these PEG derivatives all have common limitation：It can not be in usual numerous lysine residues present on protein surface Between carry out selective installation.It is more important (such as being present in enzyme active sites) for protein active in lysine residue In the case of or worked in the interaction of mediating protein and other biomolecule (such as in acceptor knot in lysine residue In the case of closing site) in the case of, this is probably an important limitation.
The complex situations of another no less important of the existing method of protein PEGylation are that PEG derivatives may The unwanted side reaction that experience occurs with the residue in addition to required residue.Histidine contains reactive imino moiety (its Representation is-N (H) -), but many and ε-NH2The chemical reactivity material of reaction also can be with-N (H)-reaction.Similarly, ammonia The side chain of base acid cysteine carries free sulfhydryl group, and its representation is-SH.In some cases, for the ε-NH of lysine2 The PEG derivatives of group also react with cysteine, histidine or other residues.This can produce bioactivity derived from PEG The complicated heterogeneous mixture of molecule, and there is the active risk of destruction institute target biology bioactive molecule.Exploitation will be needed Go out permission introduces chemical functional group PEG derivatives at the single site in protein, it will then cause one kind or a kind of Above PEG polymer can be with the bioactive molecule selectivity at determination on protein surface and predictable specific site Coupling.
Sizable effort has also been made in addition to lysine residue, in art to develop the other amino acid sides of targeting The activated PEG reagents of chain (including cysteine, histidine and N-terminal).For example, referring to being incorporated herein by reference U.S. Patent No. 6,610,281, and " Polyethylene Glycol and Derivatives for AdvancedPEGylation ", Nektar Molecular Engineering Catalog, 2003, the 1-17 pages.It can be used Cysteine residues site selectivity is introduced into protein structure by known other technologies in direct mutagenesis and art, And gained free sulfhydryl group part can be with the PEG derivatives reactions with thiol-reactive functional group.However, this method is answered Miscellaneous part is that introducing free sulfhydryl group may make gained protein expression, folding and stability become complicated.So it would be desirable to A kind of method that chemical functional group is introduced into FGF-21 is obtained, it enables one or more kinds of PEG polymer and egg White matter is selectively coupled, while compatible with other chemical functional groups typically seen in sulfydryl and protein (that is, will not can occur Unwanted side reaction).
From the point of view of the sampling in art, developing for connecting protein side chain (specifically, lysine ammonia - NH on base acid side chain2Part and cysteine side chain on-SH parts) these derivatives in, have proven to many derivatives Synthesis and using there is problem.Some derivatives form unstable bonded with protein, and it undergoes hydrolysis and therefore decomposed, drop Solve or to be otherwise in aqueous environments (such as in blood flow) unstable.It is bonded that the formation of some derivatives is relatively stablized, but Formed it is described it is bonded before undergo hydrolysis, this represent PEG derivatives on reactive group may can connect protein it It is preceding to have inactivated.Some derivatives slightly toxicity and therefore it is not particularly suitable in vivo using.Some derivatives because reacted it is slow and Can not actual use.Some derivatives cause protein active to be lost because being connected with the site of responsible protein active.Some Derivative does not have specificity in its site by connection, loss of activity needed for this may also cause and the reproduction for lacking result Property.For overcome with the challenge of polyalkylene glycol moiety modified protein qualitative correlation, developed it is more stable (for example, United States Patent (USP) 6, 602,498, it is incorporated herein by reference) or with the thiol moiety selective reaction on molecule and surface (for example, beautiful State's patent 6,610,281, it is incorporated herein by reference) PEG derivatives.Undoubtedly need requiring in art It is in chemically inert PEG derivatives that selective reaction, which is formed before stable chemical bond in physiological environment,.
In recent years, brand new technical in reporter protein matter science, it is hopeful to overcome numerous special with protein site The related limitation of opposite sex modification.Specifically, new component is added to prokaryotes Escherichia coli (Escherichia coli；E.coli) (for example, L.Wang et al., (2001),Science292：498-500) and eucaryote saccharomyces cerevisiae (Sacchromyces cerevisiae；S.cerevisiae) (for example, J.Chin et al.,Science301：964-7 (2003) in Protein synthesis mechanism), it makes it possible to that non-genetic coding amino acid in vivo is incorporated into protein In.Using the method, responded amber codon TAG and by many novel ammonia with novel chemistry, physics or biological property Base acid (including photoaffinity mark and can photoisomerization amino acid, photo-crosslinking amino acid (referring for example to Chin, J.W. et al., (2002)Proc.Natl.Acad.Sci.U.S.A, 99：11020-11024；And Chin, J.W. et al., (2002)J.Am.Chem.Soc.124：9026-9027), ketone group amino acid, amino acid containing heavy atom and glycosylated amino acid) it is effectively and high It is incorporated to high fidelity in the protein in Escherichia coli and yeast.For example, referring to J.W.Chin et al., (2002),Journal of the American Chemical Society124：9026-9027；J.W.Chin and P.G.Schultz, (2002),ChemBioChem3(11)：1135-1137；J.W.Chin et al., (2002),PNAS United States of America99：11020-11024；And L.Wang and P.G.Schultz, (2002),Chem.Comm.,1：1-11.All ginsengs It is all to be incorporated herein in entirety by reference to examine document.These researchs have proven to be possible to optionally and routinely to draw Enter non-existent chemical functional group's (such as ketone group, alkynyl and azide moiety) in protein, the functional group is common to 20 kinds All functional groups present in genetic coding amino acid are formed all in chemical inertness and available for effectively and selectively reaction It is stable covalently bonded.
Naturally occurring functional group may be provided by the ability of non-genetic coding amino acid is incorporated in protein allowing to introduce (ε-the NH of such as lysine2, cysteine sulfydryl-SH, the imino group of histidine etc.) valuable replacement chemical function Group.Functional group present in known some chemical functional groups genetic coding amino acid common to 20 kinds is inert, but completely simultaneously Effectively reaction formation stablizes bonded.For example, known azido and acetenyl exist in the copper of catalytic amount in art Under undergo Hughes's root (Huisgen) [3+2] cycloaddition reaction under aqueous conditions.For example, referring to Tornoe et al., (2002)J.Org.Chem.67：3057-3064；With Rostovtsev et al., (2002)Angew.Chem.Int.Ed.41：2596- 2599.For example, by introducing azide moiety in protein structure, can be incorporated to amine present in protein, sulfydryl, Carboxylic acid, hydroxyl are in chemical inertness, but also steady and effectively react and form the functional group of cycloaddition product with acetylene moiety.It is important , in the case of in the absence of acetylene moiety, azide is still protected in physiological conditions in the presence of other oroteins side chain Hold chemical inertness and do not react.
The invention particularly relates to the activity to FGF-21 polypeptides and the problem of prepares related, and it is directed to have what is improved The preparation of the FGF-21 polypeptides of biological or pharmacological property (the treatment half-life period of such as improvement).
The content of the invention
The present invention provides the FGF-21 polypeptides for including one or more non-naturally encoded amino acids.
In certain embodiments, FGF-21 polypeptides include one or more posttranslational modifications.In certain embodiments, FGF-21 polypeptides are connected with connexon, polymer or bioactive molecule.In certain embodiments, FGF-21 polypeptides be with it is double Functional polymer, bifunctional linker or the connection of at least one other FGF-21 polypeptides.
In certain embodiments, non-naturally encoded amino acids are connected with water-soluble polymer.In certain embodiments, water Soluble polymer includes polyalkylene glycol moiety.In certain embodiments, non-naturally encoded amino acids be using connexon with it is water-soluble Property polymer connection or with water-soluble polymer be bonded.In certain embodiments, peg molecule is double functional copolymer. In some embodiments, double functional copolymer is connected with the second polypeptide.In certain embodiments, the second polypeptide is FGF-21 many Peptide.
In certain embodiments, FGF-21 polypeptides include at least two and the water-soluble polymer comprising polyalkylene glycol moiety The amino acid of connection.In certain embodiments, at least one amino acid is non-naturally encoded amino acids.
In certain embodiments, one or more non-naturally encoded amino acids are incorporated in FGF-21 following position In one or more positions：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14, 15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、 40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、 65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、 90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、 111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、 130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、 149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、 168th, 169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl of protein End) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one or one with Upper non-naturally encoded amino acids are incorporated to SEQ ID NO：In 34-36 before position 1 (that is, in N-terminal) to one of C-terminal or In more than one position.In certain embodiments, one or more non-naturally encoded amino acids are incorporated to the following of FGF-21 In one or more positions in position：10、52、117、126、131、162、87、77、83、72、69、79、91、96、 108 and 110 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one or one Individual above non-naturally encoded amino acids are incorporated in one or more positions in FGF-21 following position：10、52、77、 117、126、131、162(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one Individual or more than one non-naturally encoded amino acids are incorporated in one or more positions in FGF-21 following position：87、 77、83、72(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one or one Individual above non-naturally encoded amino acids are incorporated in one or more positions in FGF-21 following position：69、79、91、 96th, 108 and 110 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one or More than one alpha-non-natural amino acid is incorporated to SEQ ID NO：3rd, the targeting sequencing or signal sequence of 4,6,7 or other FGF-21 sequences In.In certain embodiments, targeting sequencing may be selected from SEQ ID NO：39th, 40,41,42,43 or 44.In some embodiments In, FGF-21 secretions construct is cloned into selected from SEQ ID NO：39th, 40,41,42,43,44 targeting sequencing In pVK7ara (Nde/Eco).
In certain embodiments, by the non-naturally-occurring amino acid and water-soluble poly at one or more described positions Compound is connected, including but not limited to following position：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10, 11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、 36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、 61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、 86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、 108th, 109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126, 127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、 146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、 165th, 166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, exist The carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, By SEQ ID NO：To the non-day at one or more positions of C-terminal before position 1 (that is, in N-terminal) in 34-36 So there is amino acid to be connected with water-soluble polymer.In certain embodiments, will be non-at one or more described positions Naturally occurring amino acid is connected with water-soluble polymer, including but not limited to following position：10、52、117、126、131、 162nd, 87,77,83,72,69,79,91,96,108 and 110 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino in 2-7 Acid).In certain embodiments, by the non-naturally-occurring amino acid and water-soluble polymer at one or more described positions Connection, including but not limited to following position：10、52、77、117、126、131、162(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, by the non-naturally-occurring ammonia at one or more described positions Base acid is connected with water-soluble polymer：87、77、83、72(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino in 2-7 Acid).In certain embodiments, by the non-naturally-occurring amino acid and water-soluble polymer at one or more described positions Connection：69th, 79,91,96,108 and 110 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).At some In embodiment, by SEQ ID NO：3rd, 4,6,7,39,40,41,42,43,44 targeting sequencing or signal sequence or other One or more non-naturally-occurring amino acid in FGF-21 sequences are connected with water-soluble polymer.In some embodiments In, by SEQ ID NO：3rd, one or one in 4,6,7 targeting sequencing or signal sequence or other FGF-21 sequences with Upper non-naturally-occurring amino acid is connected with water-soluble polymer.
In certain embodiments, FGF-21 polypeptides are comprising regulation FGF-21 polypeptides are to FGF-21 polypeptide receptors or combine collocation Substitution, addition or the missing of the compatibility of thing (including but not limited to protein, polypeptide, small molecule or nucleic acid).In some realities Apply in example, FGF-21 polypeptides are included increases FGF- compared with the stability of the corresponding FGF-21 without substitution, addition or missing Substitution, addition or the missing of the stability of 21 polypeptides.In certain embodiments, FGF-21 polypeptides include with without replacing, add Plus or missing corresponding FGF-21 immunogenicity compared to regulation FGF-21 polypeptides immunogenicity substitution, addition or lack. In certain embodiments, FGF-21 polypeptides include the serum half-life to the corresponding FGF-21 without substitution, addition or missing Or circulation time is compared to the serum half-life of regulation FGF-21 polypeptides or the substitution of circulation time, addition or lacks.
In certain embodiments, FGF-21 polypeptides include the water to the corresponding FGF-21 without substitution, addition or missing Dissolubility is compared to the water miscible substitution of increase FGF-21 polypeptides, addition or missing.In certain embodiments, FGF-21 polypeptides are included Increase the FGF-21 polypeptides produced in host cell compared with the dissolubility of the corresponding FGF-21 without substitution, addition or missing Deliquescent substitution, addition or missing.In certain embodiments, FGF-21 polypeptides include with without substitution, addition or lack The corresponding FGF-21 lost expression or synthesis is in vitro closed compared to expression or increase of the FGF-21 polypeptides in host cell is increased Into substitution, addition or lack.Agonist activity is kept comprising this substituted FGF-21 polypeptides and is kept or is improved in place Expression in chief cell.In certain embodiments, FGF-21 polypeptides include corresponding to without what is replaced, add or lack FGF-21 protease resistant is compared to the substitution of the protease resistant of increase FGF-21 polypeptides, addition or lacks.U.S. Patent No. No. 6,716,626 point out to be substituted with change the potential site of protease cracking include but is not limited to 2 of proline it is residual Single alkali site in base.In certain embodiments, FGF-21 polypeptides include to without substitution, addition or missing it is corresponding The activity of acceptor is compared to the substitution of the signal transduction activity of regulation FGF-21 acceptors, addition after many peptide interactions of FGF-21 or lacks Lose.In certain embodiments, FGF-21 polypeptides are included and without substitution, addition or the combination of the corresponding FGF-21 polypeptides lacked Substitution, addition or missing compared to the combination for adjusting itself and another molecule (such as acceptor).
In certain embodiments, FGF-21 polypeptides include the phase to the corresponding FGF-21 without substitution, addition or missing Substitution, addition of the capacitive compared to increase FGF-21 polypeptides with the compatibility of medical preservative (for example, metacresol, phenol, benzylalcohol) Or missing.This increased compatibility will make it possible to prepare the physiochemical properties and biology of the holding protein during storing The anti-corrosion pharmaceutical formulation of activity.The WO 2005/091944 being incorporated herein in entirety by reference is discussed with enhanced The following instance of the FGF-21 mutains of medical stability：With powered and/or polarity but uncharged 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor one Following amino acid：WO 05/091944 SEQ ID NO：1 glycine 42, glutamine 54, arginine 77, alanine 81, Leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126, proline 130, arginine 131, leucine 139th, alanine 145, leucine 146, isoleucine 152, alanine 154, glutamine 156, glycine 161, serine 163rd, glycine 170 or serine 172.The FGF-21 polypeptides of the present invention may include one of corresponding position in polypeptide or More than one described substitution, and/or may include one or more other substitutions, addition or lack.In certain embodiments, One or more alpha-non-natural amino acids are replaced at one or more positions in following position：Glycine 42nd, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122, histidine 125, smart ammonia Acid 126, proline 130, arginine 131, leucine 139, alanine 145, proline/leucine 146, isoleucine 152, third Propylhomoserin 154, glutamine 156, glycine 161, serine 163, glycine 170, (the SEQ IDNO of serine 172：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one or more alpha-non-natural amino acids are in following position In one or more positions at replaced：Glutamic acid 91, arginine 131, glutamine 108, arginine 77, smart ammonia Acid 72, histidine 87, leucine 86, arginine 126, glutamic acid 110, tyrosine 83, proline 146, arginine 135, smart ammonia Acid 96, (the SEQ ID NO of arginine 36：1 or in SEQ IDNO：Corresponding amino acid in 2-7).
Other FGF-21 mutains of the descriptions of WO 05/091944 with enhanced medical stability.These mutains Including with cysteine replace FGF-21 in two or more following amino acid (referring to WO 05/091944 SEQ ID NO：1)：Arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartic acid 24, aspartic acid 25, the third ammonia It is acid 26, glutamine 27, glutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, sweet Propylhomoserin 42, glycine 43, glutamic acid 50, glutamine 54, leucine 58, valine 62, leucine 66, glycine 67, bad ammonia It is acid 69, arginine 72, phenylalanine 73, glutamine 76, arginine 77, aspartic acid 79, glycine 80, alanine 81, bright Propylhomoserin 82, glycine 84, serine 85, proline 90, alanine 92, serine 94, phenylalanine 95, leucine 100, asparagus fern Propylhomoserin 102, tyrosine 104, tyrosine 107, serine 109, glutamic acid 110, proline 115, histidine 117, leucine 118th, proline 119, asparagine 121, lysine 122, serine 123, proline 124, histidine 125, arginine 126, Aspartic acid 127, alanine 129, proline 130, glycine 132, alanine 134, arginine 135, leucine 137, dried meat ammonia Acid 138 or leucine 139.The FGF-21 polypeptides of the present invention may include one or more of the corresponding position in polypeptide The substitution, and/or may include one or more other substitutions, addition or lack.In certain embodiments, one or one Individual above alpha-non-natural amino acid is replaced at one or more positions in following position：Arginine 19, tyrosine 20th, leucine 21, tyrosine 22, threonine 23, aspartic acid 24, aspartic acid 25, alanine 26, glutamine 27, paddy ammonia Acid amides 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, paddy ammonia Acid 50, glutamine 54, leucine 58, valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylpropyl alcohol ammonia Acid 73, glutamine 76, arginine 77, aspartic acid 79, glycine 80, alanine 81, leucine 82, glycine 84, silk ammonia Acid 85, proline 90, alanine 92, serine 94, phenylalanine 95, leucine 100, aspartic acid 102, tyrosine 104, junket Propylhomoserin 107, serine 109, glutamic acid 110, proline 115, histidine 117, leucine 118, proline 119, asparagine 121st, lysine 122, serine 123, proline 124, histidine 125, arginine 126, aspartic acid 127, alanine 129, Proline 130, glycine 132, alanine 134, arginine 135, leucine 137, proline 138 or (the SEQ ID of leucine 139 NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
WO 05/091944 is further described also has engineering in addition to the naturally occurring disulfide bond at Cys75-Cys93 The specific FGF-21 mutains (amino acid replaced through cysteine) for the disulfide bond transformed, it is as follows：Gln76Cys- Ser109Cys、Cys75-Ser85Cys、Cys75-Ala92Cys、Phe73Cys-Cys93、Ser123Cys-His125Cys、 Asp102Cys-Tyr104Cys、Asp127Cys-Gly132Cys、Ser94Cys-Glu110Cys、Pro115Cys- His117Cys、Asn121Cys-Asp127Cys、Leu100Cys-Asp102Cys、Phe95Cys-Tyr107Cys、 Arg19CysPro138Cys、Tyr20Cys-Leu139Cys、Tyr22Cys-Leu137Cys、Arg77Cys-Asp79Cys、 Pro90Cys-Ala92Cys、Glu50Cys-Lys69Cys、Thr23Cys-Asp25Cys、Ala31Cys-Gly43Cys、 Gln28Cys-Gly43Cys、Thr23Cys-Gln28Cys、Val41Cys-Leu82Cys、Leu58Cys-Val62Cys、 Gln54Cys-Leu66Cys、Ile35Cys-Gly67Cys、Gly67Cys-Arg72Cys、Ile35Cys-Gly84Cys、 Arg72Cys-Gly84Cys or Arg77Cys-Ala81Cys, wherein numbering is the SEQ ID NO according to WO 05/091944：1. The mutain of other disulfide bond with engineered mistake is Tyr22Cys-Leu139Cys；Asp24Cys-Arg135Cys； Leu118Cys-Gly132Cys；His117Cys-Pro130Cys；His117Cys-Ala129Cys；Leu82Cys- Pro119Cys；Gly80Cys-Ala129Cys；Gly43Cys-Pro124Cys；Gly42Cys-Arg126Cys；Gly42Cys- Pro124Cys；Gln28Cys-Pro124Cys；Gln27Cys-Ser123Cys；Ala26Cys-Lys122Cys；Or Asp25Cys-Lys122Cys, wherein numbering is the SEQ ID NO according to WO 05/091944：1.It is other that there is engineered mistake Disulfide bond mutain be Leu118Cys-Ala134Cys；Leu21Cys-Leu33Cys；Ala26Cys-Lys122Cys； Leu21Cys-Leu33Cys/Leu118Cys-Ala134Cys, wherein numbering is the SEQ ID NO according to WO05/091944：1. The FGF-21 polypeptides of the present invention may include one or more described substitutions of the corresponding position in polypeptide, and/or can Including one or more other substitutions, addition or missings.The FGF-21 polypeptides of the present invention may include corresponding in polypeptide One or more described substitution (SEQ ID NO at position：1 or in SEQ ID NO：Corresponding amino acid in 2-7). In some embodiments, FGF-21 polypeptides of the invention may include in SEQ ID NO：In 34-36 from position 1 (that is, in N-terminal) it One or more described substitutions of the preceding corresponding position to C-terminal.
WO 05/091944 describes other FGF-21 mutains of Pegylation.These mutains have it is a kind of with Lower substitution：D25C、D38C、L58C、K59C、P60C、K69C、D79C、H87C、E91C、E101C、D102C、L114C、L116C、 K122C、R126C、P130C、P133C、P140C.The FGF-21 polypeptides of the present invention may include the corresponding position in polypeptide One or more described substitutions, and/or may include one or more other substitutions, addition or lack.In some realities Apply in example, one or more alpha-non-natural amino acids are replaced at one or more positions in following position： 25、38、58、59、60、69、79、87、91、101、102、114、116、122、126、130、133、140(SEQ ID NO：1 or In SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, FGF-21 polypeptides of the invention may include in SEQ ID NO：To being taken described in one or more of the corresponding position of C-terminal before position 1 (that is, in N-terminal) in 34-36 Generation.
WO 05/091944 describes the cysteine substitution at following position：19、21、26、28、29、30、36、39、 42nd, 50,56,61,64,65,68,70,71,77,81,85,86,90,92,94,98,107,108,112,113,123 and 124. WO 05/091944 illustrates the cysteine substitution at following position：24、27、37、40、44、46、49、57、88、89、106、 110th, 111,115,120 and 139.WO 05/091944 is also described in the cysteine substitution at following position：18、45、47、 48th, 78,83,99,103,125,128,131,132 and 138.WO 05/091944 is also described in the cysteine at following position Substitution：25th, 38,58,59,60,69,79,87,91,101,102,114,116,122,126,130,133 and 140.
In certain embodiments, the key of one or more engineered mistakes is by one or more non-natural ammonia Base acid is produced.The key of intramolecular can be produced in many ways, two amino acid (one or two including but not limited in protein Individual amino acid may be alpha-non-natural amino acid) reaction under suitable conditions；(it may be each natural coding to two amino acid Or non-naturally encoded) and connexon, the reaction of polymer or other molecules under suitable conditions etc..
In certain embodiments, one or more amino acid in FGF-21 polypeptides can be through one or more days So exist or non-naturally-occurring 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.In certain embodiments, the amino acid in FGF-21 polypeptides can be through naturally occurring Or non-naturally-occurring 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, condition is that at least one replaces through non-naturally encoded amino acids.In certain embodiments, One or more amino acid in FGF-21 polypeptides can be through one or more naturally occurring 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and separately At least one replaces through non-naturally encoded amino acids outside.
In certain embodiments, non-naturally encoded amino acids include carbonyl, acetyl group, aminooxy group, diazanyl, hydrazide group, ammonia Base urea groups, azido or alkynyl.
In certain embodiments, non-naturally encoded amino acids include carbonyl.In certain embodiments, non-naturally encoded amino Acid has following structure：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl or substituted aryl；R2For H, alkyl, aryl, through taking Substituted alkyl and substituted aryl；And R3Base, and R are modified for H, amino acid, polypeptide or amino terminal4For H, amino acid, polypeptide Or carboxyl terminal modification base.
In certain embodiments, non-naturally encoded amino acids include aminooxy group.In certain embodiments, non-naturally encoded ammonia Base acid includes hydrazide group.In certain embodiments, non-naturally encoded amino acids include diazanyl.In certain embodiments, non-natural Coded amino acid residue includes amino urea groups.
In certain embodiments, non-naturally encoded amino acids residue includes azido.In certain embodiments, non-natural is compiled Code amino acid has following structure：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl, substituted aryl or it is not present；X is for O, N, S or not In the presence of；M is 0-10；R2Base, and R are modified for H, amino acid, polypeptide or amino terminal3For H, amino acid, polypeptide or carboxyl terminal Modify base.
In certain embodiments, non-naturally encoded amino acids include alkynyl.In certain embodiments, non-naturally encoded amino Acid has following structure：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl or substituted aryl；X is O, N, S or is not present；M is 0-10；R2Base, and R are modified for H, amino acid, polypeptide or amino terminal3Base is modified for H, amino acid, polypeptide or carboxyl terminal.
In certain embodiments, polypeptide be FGF-21 polypeptide agonists, partial agonist, antagonist, partial antagonist or Inverse agonist.In certain embodiments, FGF-21 polypeptide agonists, partial agonist, antagonist, partial antagonist or reverse Activator includes the non-naturally encoded amino acids being connected with water-soluble polymer.In certain embodiments, water-soluble polymer bag Containing polyalkylene glycol moiety.In certain embodiments, FGF-21 polypeptide agonists, partial agonist, antagonist, partial antagonist or Inverse agonist includes non-naturally encoded amino acids and one or more posttranslational modifications, connexon, polymer or life Thing bioactive molecule.
The present invention also provide comprising under strict conditions with SEQ ID NO：The seperated nuclear acid of the polynucleotides of 8-14 hybridization. The present invention also provide comprising under strict conditions with SEQ ID NO：The seperated nuclear acid of the polynucleotides of 8-14 hybridization, wherein described Polynucleotides include at least one selection codon.The present invention is also provided comprising coding with SEQ ID NO：The polypeptide that 1-7 is represented Polynucleotides seperated nuclear acid.The present invention is also provided has one or more non-naturally encoded amino acids comprising coding With SEQ ID NO：The seperated nuclear acid of the polynucleotides for the polypeptide that 1-7 is represented.Those skilled in the art is it is clear that a variety of Any polypeptide of the different polynucleotides codified present invention.
In certain embodiments, selection codon is selected from by amber codon, ochre codon, opal codon (opalcodon), the group of unique codon, rare codon, five base codons and four base codons composition.
The method that the present invention also provides the FGF-21 polypeptides that manufacture is connected with water-soluble polymer.In certain embodiments, Methods described include make the separation FGF-21 polypeptides comprising non-naturally encoded amino acids with comprising with the non-naturally encoded amino The water-soluble polymer contact of the part of acid reaction.In certain embodiments, it is incorporated to the non-naturally encoded ammonia in FGF-21 polypeptides Base acid can react with the water-soluble polymer or not reaction of any of 20 kinds of common amino acids on the contrary.In some embodiments In, be incorporated to non-naturally encoded amino acids in FGF-21 polypeptides on the contrary can with or not the reaction of any of 20 kinds of common amino acids Connexon, polymer or bioactive molecule reaction.
In certain embodiments, by making the FGF-21 polypeptides comprising the amino acid containing carbonyl and comprising aminooxy group, hydrazine, acyl The peg molecule of hydrazine or amino urea groups reacts to prepare the FGF-21 polypeptides being connected with water-soluble polymer.In some implementations In example, aminooxy group, hydrazine, hydrazides or amino urea groups are to be connected by the way that acid amides is bonded with peg molecule.
In certain embodiments, by make comprising carbonyl peg molecule and include aminooxy group, hydrazine, hydrazides or The polypeptides reactive of the non-naturally encoded amino acids of amino urea groups prepares the FGF-21 polypeptides being connected with water-soluble polymer.
In certain embodiments, by making the FGF-21 polypeptides comprising the amino acid containing alkynes and the poly- second comprising azide moiety Glycol molecules react to prepare the FGF-21 polypeptides being connected with water-soluble polymer.In certain embodiments, azido or alkynyl It is to be connected by the way that acid amides is bonded with peg molecule.
In certain embodiments, by making the FGF-21 polypeptides comprising the amino acid containing azido and gathering comprising alkynyl moiety Glycol molecule reacts to prepare the FGF-21 polypeptides being connected with water-soluble polymer.In certain embodiments, azido or alkynes Base is to be connected by the way that acid amides is bonded with peg molecule.
In certain embodiments, peg molecule has the molecular weight between about 0.1kDa and about 100kDa. In some embodiments, peg molecule has the molecular weight between 0.1kDa and 50kDa.
In certain embodiments, peg molecule is branched polymers.In certain embodiments, polyethylene glycol branch gathers Each side chain of compound has the molecular weight between 1kDa and 100kDa or between 1kDa and 50kDa.
In certain embodiments, the water-soluble polymer being connected with FGF-21 polypeptides includes poly- alkane glycol moiety.At some In embodiment, the non-naturally encoded amino acids residue being incorporated in FGF-21 polypeptides includes carbonyl, aminooxy group, hydrazide group, diazanyl, ammonia Base urea groups, azido or alkynyl.In certain embodiments, the non-naturally encoded amino acids residue being incorporated in FGF-21 polypeptides is included Carbonyl moiety, and water-soluble polymer includes aminooxy group, hydrazides, hydrazine or semicarbazide moiety.In certain embodiments, it is incorporated to Non-naturally encoded amino acids residue in FGF-21 polypeptides includes alkynyl moiety, and water-soluble polymer includes azide moiety. In some embodiments, the non-naturally encoded amino acids residue being incorporated in FGF-21 polypeptides includes azide moiety, and water-soluble poly Compound includes alkynyl moiety.
The present invention also provides composition, and it includes the FGF-21 polypeptides of non-naturally encoded amino acids and pharmaceutically may be used The supporting agent of receiving.In certain embodiments, non-naturally encoded amino acids are connected with water-soluble polymer.
The present invention also provides the cell of the polynucleotides comprising FGF-21 polypeptide of the coding comprising selection codon.At some In embodiment, the cell, which is included, to be used to substituting onto non-naturally encoded amino acids into the orthogonal RNA synzyme in FGF-21 polypeptides And/or orthogonal tRNA.
The method that the present invention also provides FGF-21 polypeptide of the manufacture comprising non-naturally encoded amino acids.In some embodiments In, it is many comprising one or more kinds of coding FGF-21 that methods described is included in culture under conditions of allowing FGF-21 expression of polypeptides The cell of the polynucleotides of peptide, orthogonal RNA synzyme and/or orthogonal tRNA；And purify FGF-21 from cell and/or culture medium Polypeptide.
The present invention also provides treatment half-life period, serum half-life or the method for circulation time of increase FGF-21 polypeptides.This The method that invention also provides the immunogenicity of regulation FGF-21 polypeptides.In certain embodiments, methods described includes and uses non-natural Coded amino acid replaces any one or more than one amino acid in naturally occurring FGF-21 polypeptides and/or by FGF-21 polypeptides It is connected with connexon, polymer, water-soluble polymer or bioactive molecule.
Also the method that the FGF-21 molecular therapies of the invention of offer effective dose of the invention need the patient of this treatment. In certain embodiments, methods described includes the medical composition to patient's administration therapeutically effective amount, the medical composition bag Containing FGF-21 polypeptides and pharmaceutically acceptable supporting agent including non-naturally encoded amino acids.In certain embodiments, non-day Right coded amino acid is connected with water-soluble polymer.
The present invention is also provided and included with SEQ ID NO：Sequence that 1-7 is represented or any other FGF-21 peptide sequences FGF-21 polypeptides, but wherein at least one amino acid is replaced by non-naturally encoded amino acids.The present invention is also provided and included with SEQ ID NO：1st, the FGF-21 polypeptides of 2,4 and 5 sequences represented.In certain embodiments, non-naturally encoded amino acids and water solubility Polymer is connected.In certain embodiments, water-soluble polymer includes polyalkylene glycol moiety.In certain embodiments, non-natural Coded amino acid includes carbonyl, aminooxy group, hydrazide group, diazanyl, amino urea groups, azido or alkynyl.
The present invention also provides medical composition, and it is comprising pharmaceutically acceptable supporting agent and comprising with SEQ ID NO：1- The FGF-21 polypeptides of 7 sequences represented or any other FGF-21 peptide sequences, wherein at least one amino acid is through non-natural Coded amino acid replaces.The present invention also provides medical composition, and it is comprising pharmaceutically acceptable supporting agent and comprising with SEQ ID NO：The FGF polypeptides for the sequence that 1-7 is represented.In certain embodiments, non-naturally encoded amino acids include sugar moieties.At some In embodiment, water-soluble polymer is connected by sugar moieties with polypeptide.In certain embodiments, connexon, polymer or biology Bioactive molecule is connected by sugar moieties with FGF-21 polypeptides.
The FGF-21 that the present invention also provides the water-soluble polymer connected at single amino acid by covalent bond is more Peptide.In certain embodiments, water-soluble polymer includes polyalkylene glycol moiety.In certain embodiments, with water-soluble polymer The amino acid of covalent attachment is non-naturally encoded amino acids present in polypeptide.
The present invention provides the FGF-21 polypeptides for including at least one connexon, polymer or bioactive molecule, wherein institute State the official that connexon, polymer or bioactive molecule are the non-naturally encoded amino acids by being incorporated to via ribosomes in polypeptide It can roll into a ball and be connected with polypeptide.In certain embodiments, polypeptide is through single Pegylation.The present invention is also provided and included and one or one The FGF-21 polypeptides of connexon, polymer or bioactive molecule that individual above non-naturally encoded amino acids are connected, wherein described Non-naturally encoded amino acids are to be incorporated in preselected sites via ribosomes in polypeptide.
Include the FGF-21 targeting sequencings or signal sequence being connected with FGF-21 code areas, Yi Jiyu in the scope of the invention The Heterologous signal sequences of FGF-21 code areas connection.Selected heterologous leader sequence or signal sequence should be identification and treated sequence Row, for example, secreted and may be cracked by host cell via signal peptidase by host cell excretory system.The leading sequence of the present invention Row may be selected from following：SEQ ID NO：3 and SEQ ID NO：The targeting sequencing (amino acid position 1-28) of 6 three leucines, SEQ ID NO：4 and SEQ ID NO：Targeting sequencing (amino acid position 1-27), the SEQ ID NO of 7 two leucines：2 His labels (amino acid position 1-10), SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、 SEQ ID NO：43、SEQ ID NO：44.Meant with the FGF-21 treatment symptom of the present invention or the method for illness with having or not FGF-21 treatments with signal peptide or leader peptide.
The present invention also provides the increased method of glucose uptake in inducing adipocyte, and methods described is included to described thin Born of the same parents' administration effectively can absorb the FGF-21 of increased amount by induced glucose.The glucose uptake increase can be by faster and more Effective glucose utilization and the increase for causing energy expenditure.
In another embodiment, due to being reacted for the distinct chemical for engaging alpha-non-natural amino acid, one or one is included The engagement of the FGF-21 polypeptides of above non-naturally-occurring amino acid and another molecule (including but not limited to PEG) provides substantial Purified FGF-21.It may be performed in the case of using other purification techniques non-naturally encoded comprising one or more The FGF-21 of amino acid and another molecule (such as PEG) engagement, wherein purification technique is entered before or after engagement step OK, to provide substantially pure FGF-21.
Brief description of the drawings
Fig. 1：Show the amber mutation in the corresponding site in FGF-21 and FGF-19.
Fig. 2：Show mankind FGF-19 structure.
Fig. 3：Show the amber mutation in the corresponding site in FGF-21 and FGF-2.
Fig. 4：Show mankind FGF-19 structure.
Fig. 5：Show the FGF-21 of N-terminal His marks expression and the suppression at 7 amber sites.
Fig. 6：Show the BPER supernatants of the suppression at expression and 7 amber sites from the N-terminal His FGF-21 marked Liquid sample.
Fig. 7 a：Calculate FGF21 variants 30K PEG-391,30K PEG-477,30K PEG-R131,30KPEG-Q108, The SigmaPlot figures of the EC50 values of HIS-FGF21 (wild type of His marks) serial dilution.
Fig. 7 b：The table of the active average loss multiple of the listed each Pegylation FGF21 variants of displaying.
Fig. 8：In the non-His flag Fs GF-21 of expression in escherichia coli SDS-PAGE analyses.
Fig. 9：(A) the SDS-PAGE analyses of FGF-21-Y83pAF elution parts；(B) un-marked FGF-21-Y83pAF The chromatogram of Q HP elutions；(C) the SDS-PAGE analyses of FGF-21-Y832pAFQ HP stripping cells.
Figure 10：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 11：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 12：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 13：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 14：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 15：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 16：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 17：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 18：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 19：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 20：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 21：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 22：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 23：Data from example 28, the pharmacokinetic property of FGF-21 compounds in rats.
Figure 24：PVK10-FGF21 carrier figures.
Figure 25：PVK10-FGF21 carrier sequences.
Figure 26 a：The serum concentration-time curves of the N-6His WT FGF21 of Three doses in rats.Through subcutaneously to big Mouse provides the single administration of tester.Every group of N=4 animal.Symbol represents the average value of measured serum-concentration, error bars table Show standard error.
Figure 26 b：With serum concentration-times of the 0.25mg/kg through N-6His WT FGF21 subcutaneous or through intravenous administration Curve.Through the single administration that tester is subcutaneously provided to rat.Every group of N=4 animal.Symbol represents measured serum-concentration Average value, error bars represent standard error.Total biological usability is about 87%.
Figure 27 a：The relation of the dosage of tester and serum-concentration under Cmax.With observation rather than theoretical value report Cmax Value.N=4 animal of each treatment group.Linear regression value is 0.59, and slope is 348.5 ± 91.22.
Figure 27 b：The dosage of tester and the relation of terminal half-life.N=4 animal of each treatment group.Due to apparent clear Except saturation (apparent saturation of clearance) is higher than 0.25mg/kg, so linear regression value can not be calculated.
Figure 27 c：The relation of dosage and serum-concentration AUC.AUC is reported with the observation for being pushed into infinitely great calculating.Each N=4 animal for the treatment of group.Linear regression value is 0.75, and slope is 1079 ± 194.1.
Figure 28 a：The serum concentration-time curves of the PP WT FGF21 of Three doses in rats.Through subcutaneously being carried to rat For the single administration of tester.Every group of N=4 animal.Symbol represents the average value of measured serum-concentration, and error bars represent mark Quasi- error.
Figure 28 b：It is bent with serum concentration-times of the 0.25mg/kg through PP WT FGF21 subcutaneous or through intravenous administration Line.Through the single administration that tester is subcutaneously provided to rat.Every group of N=4 animal.Symbol represents the flat of measured serum-concentration Average, error bars represent standard error.Total biological usability is about 65%.
Figure 29 a：The relation of the dosage of tester and serum-concentration under Cmax.With observation rather than theoretical value report Cmax Value.N=4 animal of each treatment group.Linear regression value is 0.92, and slope is 454.2 ± 42.42.
Figure 29 b：The dosage of tester and the relation of terminal half-life.N=4 animal of each treatment group.Due to apparent clear Except saturation is higher than 0.125mg/kg, so linear regression value can not be calculated.
Figure 29 c：The relation of dosage and serum-concentration AUC.AUC is reported with the observation for being pushed into infinitely great calculating.Each N=4 animal for the treatment of group.Linear regression value is 0.93, and slope is 1585 ± 137.1.
Figure 30 a：PP and the calculating latter stage with N6-His WT FGF21 compounds of the 0.5mg/kg through subcutaneous administration in rat The comparison of half-life period.The use of double tail t p values for testing (two-tailed t-test) calculating is 0.7715.Every group of N=3-4 is only moved Thing.
Figure 30 b：In rat PP with the cmax value of N6-His WT FGF21 compounds of the 0.5mg/kg through subcutaneous administration Compare.The use of the p value of double tail t measuring and calculations is 0.7652.Every group of N=3-4 animal.
Figure 30 c：In rat PP with the AUCinf's of N6-His WT FGF21 compounds of the 0.5mg/kg through subcutaneous administration Compare.The use of the p value of double tail t measuring and calculations is 0.4372.
Figure 31 a：The PK curves of the FGF21 isomers of ten kinds of Pegylation N6-His marks.
Figure 31 b：Absorption curve of the Pegylation FGF21 isomers after 0.25mg/kg hypodermic injections.
Figure 31 c：Elimination curve of the Pegylation FGF21 isomers after 0.25mg/kg hypodermic injections.
Figure 32：20kDa is compared with the pharmacokinetics of 30kDa Pegylations.
Figure 33：Through rat intravenous or through subcutaneous administration 0.25mg/kg 20KPEG-pAF91 (N6-His) FGF21 Plasma concentration time curve.To each animal administration single dose.Every group of N=4 animal.Symbol represents measured plasma concentration Average value, bar line represents standard deviation.Total biological usability is about 30%.
Figure 34：Secretions and displaying used targeting sequencing OmpA, MalE of two kinds of gel displaying FGF21 in Escherichia coli Action effect with StII is fabulous, as confirmed by the pericentral siphon release soluble part in the second gel.
Embodiment
Definition
It will be appreciated that the present invention is not limited to ad hoc approach specifically described herein, scheme, cell line, construct and reagent, and And can equally change.It will also be appreciated that term used herein is intended merely to reach the purpose of description specific embodiment, and not Intend limitation the scope of the present invention, the scope only should be any limitation as by appended claims.
Unless the context clearly indicates otherwise, otherwise as used in this paper and appended claims, singulative " one " It is " described " to refer to thing including multiple.So that it takes up a position, for example, to " FGF-21 " or " FGF-21 polypeptides " refer to be to one or More than one protein refers to, and including its equivalent etc. known to those skilled in the art.
Unless otherwise defined, otherwise all scientific and technical terminologies used herein all have and technology of the art The implication identical implication that personnel are generally understood.Although can by with specifically described herein similar or equivalent any method, device It is used for material in the practice or test of the present invention, but now only method for optimizing, device and material is been described by.
All publication and patent mentioned herein are all incorporated herein by reference, with reach description and Construct and the purpose of method that possibility described in for example described publication of announcement is used in combination with presently described invention.This Disclosure before the date of application that publication discussed in text only provides present application.It never should be interpreted that and recognize herein Inventor haves no right to shift to an earlier date the date of the disclosure due to prior inventions or any other reason.
Term " substantially purified " refers to can be substantially or substantially free of usual (i.e. natural with naturally occurring environment Cell, or restructuring produce FGF-21 polypeptides in the case of be host cell) present in protein or with naturally occurring ring The FGF-21 polypeptides of the component of protein interaction present in border.The FGF-21 polypeptide bags of cellular material can be substantially free of Include with less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, be less than about 4%th, the protein formulation of the contaminating protein less than about 3%, less than about 2% or less than about 1% (in terms of dry weight).When FGF-21 polypeptides Or its variant is when being recombinated to produce by host cell, protein can with about the 30% of dry cell weight, about 25%, about 20%, about 15%th, about 10%, about 5%, about 4%, about 3%, about 2% or about 1% or 1% exist below.When FGF-21 polypeptides or its variation Body is that protein can be with dry cell weight basis about 5g/L, about 4g/L, about 3g/L, about 2g/L, about when being recombinated to produce by host cell 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L or about 1mg/L or 1mg/ L is existed below in culture medium.Therefore, the FGF-21 polypeptides of " substantially purified " that is produced by the inventive method can have such as At least about 30% is determined by proper method (such as SDS/PAGE analyses, RP-HPLC, SEC and Capillary Electrophoresis), at least about 35%th, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% purity level, in particular at least about 75%, 80%, 85% purity level, and more particularly at least about 90% purity level, at least about 95% purity level, at least about 99% or more than 99% purity level.
" recombinant host cell " or " host cell " refer to regardless of method be used for insert (for example, directly intake, turn Lead, become known for producing other methods of recombinant host cell in f pairings or art), including exogenous polynucleotide is thin Born of the same parents.Exogenous polynucleotide can keep non-integrated vector (for example, plasmid) form, or can be incorporated into host genome.
As used herein, term " culture medium " include any culture medium that is sustainable or accommodating any host cell, it is molten Liquid, solid, semisolid or rigid support thing, the host cell include bacterial host cell, yeast host cell, insect host Cell, plant host cell, eukaryotic host cell, mammalian host cell, Chinese hamster ovary celI, prokaryotic host cell, Escherichia coli Or pseudomonad (Pseudomonas) host cell and cell inclusion.Therefore, the term, which can be covered, has grown host cell Culture medium, for example secreted the culture medium of FGF-21 polypeptides, be included in the culture medium before or after amplification step.For example, In the case where FGF-21 polypeptides are produced in the cell and host cell has been dissolved or ruptured to discharge FGF-21 polypeptides, institute Buffer solution or reagent containing host cell lysate can also be covered by stating term.
" reducing agent " as used in herein in connection with protein refolding is defined as making sulfydryl keep reducing condition and go back In original molecule or intermolecular disulfide bond any compound or material.Suitable reducing agent includes but is not limited to dithiothreitol (DTT) (DTT), 2 mercapto ethanol, dithioerythritol, cysteine, cysteamine (2- aminoothyl mercaptans) and reduced form gluathione Peptide.Those skilled in the art is it is clear that a variety of reducing agents are suitable for the method and composition of the present invention.
" oxidant " as used in herein in connection with protein refolding is defined as to move from the compound aoxidized Except any compound or material of electronics.Suitable oxidant includes but is not limited to oxidized form of glutathione, cystine, Guang Amine, oxidized dithiothreitol, oxidized form antierythrite and oxygen.Those skilled in the art is it is clear that a variety of oxidants Suitable for the method for the present invention.
Term " antidiabetic " should represent to can be used for treating, preventing any glucose metabolism disorder or its any complication (including any symptom, disease or complication specifically described herein) or otherwise reduce any medicine of its seriousness.It is anti- Rezulin includes insulin, thiazolidinedione, sulfonylureas, benzoic acid derivative, Alpha-glucosidase inhibitor etc..It can be this hair The antidiabetic of other general classes of a part for bright composition includes (term defined in quotation marks)：Official US's pharmacopeia (official United States Pharmacopoeia) or Official US's NF (officialNational Formulary) " medicine " approved in (or its any annex)；" new drug " and " novel chiral synthon " that U.S. FDA is ratified, these Term is used in United States Code No. 21 (of the United States Code of Title 21)；In the U.S. or overseas needs Any medicine (" approval medicine ") of government entity approval；Regulatory approved must be obtained with accordance with any of 21U.S.C. § 355 (a) Medicine (" regulatory approved medicine ")；As people's medication application (according to 21U.S.C. § 379 (g)) or just entering pedestrian's medication application (according to 21U.S.C. § 379 (g)) any medicament (" people's medication ").(referring to for all legal code names on this definition be all Refer to the code name on the original application date of present application.) other antidiabetics disclose in this article, and be art Known to technical staff.The anti-diabetic composition of the present invention is preferably able to make the reduction of HbAlc levels at least as used in this article 10% (compared to the change of baseline), and the anti-diabetic composition of the present invention is more specifically preferably able to as used in this article HbAlc levels are made to reduce at least 50% (compared to the change of baseline).Antidiabetic include insulin reinforcing agent, for example including (but not limited to) small molecule insulin reinforcing agent, i.e. taurine (Taurine), alpha lipoic acid (Alpha Lipoic Acid), mulberry Mulberry extract, chromium, glutamine, felwort (Enicostemma littorale Blume), Sweet Broomwort Herb (Scoparia Dulcis), tarragon (Tarragon) extract, Herba Andrographitis (Andrographis paniculata), isomalt (Isomalt), trehalose or D-MANNOSE, its secretion that can further enhance insulin or activity.
" denaturant " is defined as any compound or the material that protein reversible can be caused to deploy as used in this article. The intensity of denaturant will be determined by the property and concentration of specific denaturant.Suitable denaturant can for chaotropic agent (chaotrope), Cleaning agent, organic solvent, water miscible solvent, the combination of phosphatide or two or more reagent.Suitable chaotropic agent Including but not limited to urea, guanidine and sodium sulfocyanate.Applicable cleaning agent may include the strong cleaning agent of (but not limited to), such as 12 Sodium alkyl sulfate or APEO (such as Tween or Triton cleaning agents), Sarkosyl；Gentle nonionic detergent (for example, digitonin (digitonin))；Gentle cationic cleaning agent, such as N- ＞ 2,3- (two oleyl epoxides)-the third Base-N, N, N- trimethyl ammonium；Gentle Ionic detergents (such as sodium taurocholate or NaTDC)；Or amphoteric ion type cleaning Agent, including but not limited to DMPT (Zwittergent), 3- (3- courages amidopropyl) dimethylamino -1- propane Sulfate (CHAPS) and 3- (3- courages amidopropyl) dimethylamino -2- hydroxyl -1- propane sulfonates (CHAPSO).Can Use such as acetonitrile, low-carbon alkanol (especially C2-C4Alkanol, such as ethanol or isopropanol) or lower alkanes glycol (especially C2-C4Alkane Glycol, such as ethylene glycol) organic, water miscible solvent be used as denaturant.Phosphatide suitable for the present invention can be naturally to deposit In phosphatide, such as phosphatidyl-ethanolamine, phosphatidyl choline, phosphatidylserine and phosphatidylinositols；Or synthetic phospholipid derivative Or variant, such as two caproyl phosphatidyl cholines or two heptanoyl group phosphatidyl cholines.
As used herein, the polypeptide containing disulfide bond is converted into by " refolding " description from improper folding or deployed condition Disulfide bond natural or any process, reaction or the method for the conformation suitably folded.
As used herein, the specific polypeptide for referring to use at least two interaction between each other and so that expansion " are folded " altogether Or the polypeptide of improper folding is converted into refolding process, reaction or the method for natural, appropriate folded polypeptide.
As used herein, " FGF-21 polypeptides ", " FGF2 1 " or " FGF-21 " and its non-loigature Symbol form should include with FGF2 1 and FGF-21 analogs, FGF-21 hypotypes, FGF-21 analogies, FGF-21 fragments, heterozygosis FGF-21 albumen, its fusion protein, oligomer and polymer, homologue, glycosylation pattern variant, The peptide and protein of at least one bioactivity of variant, splicing variants and mutain, but regardless of the material Bioactivity is how, and more no matter its synthesis or manufacture method (including but not limited to restructuring (by cDNA, genomic DNA, Synthetic DNA or the nucleic acid of other forms are produced), in vitro, in vivo, the microinjection of nucleic acid molecules, synthesis, transgenosis and base Because of activation method) how.Term " FGF-21 polypeptides " and " FGF-21 " are covered comprising one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, added Plus or missing FGF-21 polypeptides.
Substitution at multiple amino acid positions in naturally occurring FGF-21 has been described.Substitution including but not limited to regulation doctor Drug stabilisation, increase agonist activity, increase protease resistant, polypeptide is changed into the substitution of antagonist etc., and covered In term " FGF-21 polypeptides " or " FGF-21 ".
FGF-21 sequences on lacking targeting sequencing, referring to SEQ ID NO herein：1、SEQ ID NO：2 and SEQ ID NO：5.On the FGF-21 sequences with targeting sequencing, referring to SEQ ID NO herein：3、SEQ IDNO：4、SEQ ID NO：6 and SEQ ID NO：7.In certain embodiments, FGF-21 polypeptides of the invention and SEQID NO：1-7 or FGF-21 Any other sequence of polypeptide is substantially consistent.FGF-21 a variety of polymorphs are differentiated.U.S. Patent Publication case Leucine or proline at same position are described in No. 20010012628 and U.S. Patent No. 6,716,626.The U.S. 1 amino acid (leucine) of displaying difference in patent the 6,716,626th and U.S. Patent Publication case the 20040259780th N-terminal targeting sequencing or signal sequence.Encode nucleic acid molecules and the expression of FGF-21 and FGF-21 polypeptides (including mutant) Method with purifying FGF-21 polypeptides is it is widely known that and including but not limited to U.S. Patent No. 6,716,626；The U.S. Patent publication the 2005/0176631st, No. 2005/0037457, No. 2004/0185494, the 2004/0259780th Number, No. 2002/0164713 and No. 2001/0012628；WO 01/36640；WO 03/011213；WO 03/059270； WO 04/110472；WO05/061712；WO 05/072769；WO 05/091944；WO 05/113606；WO 06/028595； WO06/028714；WO 06/050247；WO 06/065582；Person disclosed in WO 06/078463, the patent is in full The mode of reference is incorporated herein.
Term " FGF-21 polypeptides " also includes naturally occurring FGF-21 pharmaceutically acceptable salt and prodrug, and salt Prodrug, polymorph, hydrate, solvate, bioactive fragment, bioactivity variant and stereoisomer, Yi Jitian So there is FGF-21 and its polypeptide fusion activator, analogies and antagonist variant.Term " FGF-21 polypeptides " is covered The fusion of other amino acid is included in amino terminal, carboxyl terminal or two ends.Exemplary fusion includes (but not limiting In)：For example, making egg ammonia produced by FGF-21 mature form by shortage leader peptide or signal peptide or part thereof recombination expression (methionine and FGF-21 N-terminal connection, are produced the methinyl base FGF-21 that acid is connected with FGF-21 N-terminal by recombination expression It is raw)；Fusion formed by for purification purposes (is including but not limited to melted with polyhistidyl or compatibility epitope Close)；With the fusion of seralbumin binding peptide formation；With the fusion formed with haemocyanin (such as seralbumin). A kind of selection novel protein of U.S. Patent No. 5,750,373 (it is incorporated herein by reference) description (is for example given birth to Long hormone) and the method for antibody fragment variant that has changed to the binding property of acceptor molecule out of the ordinary.Methods described is included will The gene for encoding protein of interest is merged with the carboxyterminal domain of filobactivirus M13 gene III coat protein.It can produce Include chimeric point of FGF-21 and one or more other molecules (including but is not limited to keratinocyte growth factor (KGF)) Son (Reich-Slotky, R. et al., J.Biol.Chem.270：29813-29818(1995)).Chimeric molecule can contain FGF- 21 with the specific region of one or two kinds of molecules or fragment in KGF molecules.Can be by standard biochemical method by protein system Standby any fragment, or encode the polynucleotides of the fragment to prepare the fragment by expression.FGF-21 or its fragment The fusion protein form that human serum albumin (HSA) or part thereof can be included is produced.The fusion construct is suitable to enhancing The expression of FGF-21 or its fragment in eukaryotic host cell.Such as U.S. Patent No. 5,766,883 and PCT Publication case WO 97/ Disclosed in 24445 (it is incorporated herein by reference), exemplary HSA part include N-terminal polypeptide (amino acid/11- 369th, 1-419, and by the intermediate length amino acid/11).Other chimeric polyeptides may include with the C-terminal and N with HSA FGF-21 or the HSA albumen of its fragment that end is each connected.The HSA constructs, which are disclosed in, to be hereby incorporated herein by In U.S. Patent No. 5,876,969 in.FGF-21 mammalian cell expression, which is described in, is herein incorporated by reference this In WO 2005/091944 in text.
Multiple bibliography disclose to engage or glycosylate by polymer and polypeptide are modified.Term " FGF-21 polypeptides " Including the polypeptide engaged with the polymer such as PEG, and can comprising it is one or more kinds of by cysteine, lysine or its Other derivatives that its residue is carried out.In addition, FGF-21 polypeptides can include connexon or polymer, wherein with connexon or polymer The amino acid of engagement can be the alpha-non-natural amino acid according to the present invention, or using known technology in art (for example with Lysine or cysteine coupling) connexon or polymer is engaged with natural coded amino acid.
The polymer engagement of FGF-21 and other polypeptides is reported.For example, referring to WO 2005/091944, it is with reference Mode is incorporated herein.The lysine deletion polypeptide of 4,904, No. 584 announcement Pegylations of U.S. Patent No., wherein at least one Individual lysine residue has been lacked or by any other radical amino acid replacement.WO 99/67291, which discloses one kind, makes protein and PEG At least one amino acid residue in the method for engagement, wherein protein has lacked and has been enough to realize what is engaged with protein Under the conditions of protein is contacted with PEG.The Pegylation that WO 99/03887 discloses the polypeptide for belonging to growth hormone superfamily becomes Allosome, wherein cysteine residues are replaced by the non-essential amino acid residues in polypeptide designated area.WO 00/26354 A kind of method for the glycosylated polypeptides variant for preparing the allergenicity with reduction is disclosed, the variant and corresponding parent are more Peptide is compared and includes at least one other glycosylation site.(it is hereby incorporated herein by U.S. Patent No. 5,218,092 In) modification of the announcement to granulocyte colony stimulating factor (G-CSF) and other polypeptides, to introduce at least one other carbon hydrate Thing chain (compared with natural polypeptides).
Term " FGF-21 polypeptides " also includes glycosylation FGF-21, the sugar such as (but not limited to) at any amino acid position The N connections of the polypeptide, polypeptide of base or O connection glycoforms.The variant changed containing single nucleotides is also regarded as The bioactivity variant of FGF-21 polypeptides.In addition, also including splicing variants.Term " FGF-21 polypeptides " also includes by chemistry Mode is connected or with any one or more than one FGF-21 polypeptides or any other polypeptide, albumen of fusion protein form expression Matter, carbohydrate, polymer, small molecule, connexon, part or any kind of other bioactive molecules FGF-21 it is many Peptide heterodimer, homodimers, heteromultimer thing or homopolymeric thing, and contain such as particular hole or other modifications But still keep the polypeptide analog of bioactivity.
Unless otherwise indicated (that is, when explanation is based on SEQ ID NO：2nd, 3,4,5,6,7 or other FGF-21 sequences are compared Compared with when), it is otherwise all that SEQ IDNO are all based on to referring to for amino acid position in FGF-21 specifically described herein：Position in 1 Put.For example, SEQ ID NO：Amino acid at 1 position 77 is arginine, and corresponding arginine is located at SEQ ID NO：At position 87 in 2.It will be understood by one of ordinary skill in the art that in any other FGF-21 molecules (such as SEQ ID NO： 2nd, 3,4,5,6 and 7) in can easily differentiate corresponding to SEQ ID NO：The amino acid position of position in 1.The skill of art Art personnel in any other FGF-21 molecules (such as FGF-21 fusions, variant, fragment) it will be appreciated that can easily reflect Dui Yingyu not SEQ ID NO：1st, the amino acid position of the position in 2,3,4,5,6,7 or any other FGF-21 sequences.Citing For, the alignment programs such as BLAST can be used to compare and differentiate and SEQ ID NO：1st, 2,3,4,5,6,7 or other The specified protein position of position consistency in FGF-21 sequences.Herein in connection with SEQ ID NO：1st, 2,3,4,5,6,7 or its 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or addition described in its FGF-21 sequence are intended to refer to known in specifically described herein or art Substitution, missing or addition in the relevant position of FGF-21 fusions, variant, fragment etc., and clearly covered by the present invention.
Term " FGF-21 polypeptides " or " FGF-21 " are covered comprising one or more amino acid replacement, adding or deletions FGF-21 polypeptides.The FGF-21 polypeptides of the present invention can include the modification of one or more natural amino acids and one or one Individual above alpha-non-natural amino acid modification.It is described in exemplary in multiple amino acid positions in naturally occurring FGF-21 polypeptides Substitution, it includes but is not limited to adjust the substitution of medical stability, the one or more biology of regulation FGF-21 polypeptides Activity (such as (but not limited to) increases agonist activity, increase polypeptide dissolubility, reduces protease sensitive, converts polypeptide Into antagonist etc.) substitution, and term " FGF-21 polypeptides " covers the substitution.In certain embodiments, FGF-21 antagonisms Agent includes the non-naturally encoded amino acids being connected with water-soluble polymer present in the receptorbinding region of FGF-21 molecules.
In certain embodiments, FGF-21 polypeptides further include the addition of the bioactivity of regulation FGF-21 polypeptides, taken Generation or missing.For example, add, replace or lack adjustable FGF-21 one or more kinds of properties or activity.Citing For, addition, substitution or the adjustable compatibility to FGF-21 polypeptide receptors of missing adjust circulating half-life, adjustment for the treatment of half Decline the phase, adjust the stability of polypeptide, adjust the cracking carried out by protease, regulating dosage, regulation release or biological usability, promote Enter purifying, or improve or change specific dosing way.Similarly, FGF-21 polypeptides can include protease cleavage sequence, reactivity Group, antibody binding domain (include but is not limited to FLAG or poly-His) or other sequences based on compatibility (including but are not limited In FLAG, poly-His, GST etc.) or improvement polypeptide detection (include but is not limited to GFP), purifying or the connection of other characteristics Molecule (includes but is not limited to biotin).
Term " FGF-21 polypeptides " is also covered by connected homodimers, heterodimer, homopolymeric thing and heterogeneous Polymer, it including but not limited to passes through non-naturally encoded amino acids side chain and identical or different non-naturally encoded amino acids Side chain and natural encoded amino acid side chain are directly connected to or by the connexon person of being indirectly connected with.Exemplary connexon is included (but not It is limited to) small organic compound, the water-soluble polymer (such as polyethylene glycol or dextran) of various length or various length Polypeptide.
" non-naturally encoded amino acids " refer in not 20 kinds of common amino acids or pyrrolysine or selenocysteine A kind of amino acid.Can with term " non-naturally encoded amino acids " the synonymous other terms used be " alpha-non-natural amino acid ", " non-naturally-occurring amino acid ".Term " non-naturally encoded amino acids " is also including but not limited to by modification (such as after translating Modification) natural coded amino acid (including but not limited to 20 kinds of common amino acids or pyrrolysine and selenocysteine) and In the presence of, but itself can not be by translating the amino acid that compound is naturally incorporated in the polypeptide chain grown.These non-naturals are deposited Include but is not limited to N- acetyl glucosamines base-Serine, N- acetyl glucosamine bases-L- Soviet Unions ammonia in the example of amino acid Acid and O- phosphotyrosines.
" amino terminal modification base " refers to any molecule that can be connected with the amino terminal of polypeptide.Similarly, " carboxyl terminal Modification base " refers to any molecule that can be connected with the carboxyl terminal of polypeptide.End modified base includes but is not limited to various water-soluble Property polymer, peptide or protein matter (such as seralbumin), or increase peptide serum half-life other parts.
Term " functional group ", " active part ", " activated group ", " leaving group ", " reactive site ", " chemical reactivity Group " and " chemical reaction part " be in art and herein for refer to it is unique in molecule can characterizing portion or list Member.These terms are synonymous to a certain extent in chemical field, and herein be indicated for perform certain function or Activity and the molecular moiety that can be reacted with other molecules.
Term " bonded " or " connexon " are typically due to chemically react and formed and usually common for finger herein The group or key of valence link connection.Hydrolysis-stable is bonded mean it is described bonded substantially stable in water and under applicable pH value (including but is not limited in physiological conditions), (perhaps even indefinite duration) did not reacted with water for a long time.Hydrolytically unstable is degradable Bonded mean described bonded can be degraded in water or the aqueous solution (including such as blood).The unstable or degradable key of enzymatic Connection means described bonded can be degraded by one or more kinds of enzymes.It will be appreciated that PEG and related polymer are poly- in art Linking group in compound main chain or between one or more functional end-groups of main polymer chain and polymer molecule In may include it is degradable bonded.For example, institute is reacted with the alcohol radical on bioactivator by PEG carboxylic acids or activation PEG carboxylic acids The ester bond UNICOM of formation often hydrolyzes to discharge the bioactivator in physiological conditions.Other hydrolysis it is degradable it is bonded including (but not limited to) carbonic ester is bonded；Imines as produced by amine with aldehyde reaction is bonded；React what is formed with phosphate-based by alcohol Phosphate is bonded；Join as the hydrazone key of hydrazides and the reaction product of aldehyde；Acetal as aldehyde and the reaction product of alcohol is bonded；As The ortho esters of the reaction product of formates and alcohol is bonded；Amido by the including but not limited to end of the polymer such as PEG Peptide with the carboxyl formation of peptide is bonded；And by including but is not limited to the phosphoramidite of polymer ends (phosphoramidite) oligonucleotides of the 5 ' hydroxyls formation of base and oligonucleotides is bonded.
Term " bioactive molecule ", " biologically-active moiety " or " bioactivator " with this article when mean can Influence with organism (include but is not limited to virus, bacterium, bacteriophage, transposon, prion, insect, fungi, plant, animal with And the mankind) relevant biosystem, path, any material of molecule or any physics of interaction or biochemical property. Specifically, bioactive molecule as used in this article includes but is not limited to be intended for use in diagnosing, cures, alleviates, treating Or the disease or body or mental health conditions for otherwise strengthening the mankind or animal of the prevention mankind or other animals Any material.The example of bioactive molecule includes but is not limited to peptide, protein, enzyme, small-molecule drug, vaccine, immune Former, hard medicine (hard drug), soft medicine (soft drug), carbohydrate, inorganic atoms or molecule, dyestuff, lipid, nucleosides, Radionuclide, oligonucleotides, toxoid, toxin, protokaryon and eukaryotic, virus, polysaccharide, from virus, bacterium, insect, dynamic Thing or any other cell or cell type, liposome, particulate and micella are obtained or obtained nucleic acid and its part.Suitable for this The species of the bioactivator of invention includes but is not limited to medicine, prodrug, radionuclide, developer, polymer, antibiosis Element, fungicide, antivirotic, antiphlogistic, antineoplastic, cardiovascular drug, anxiolytic, hormone, growth factor, steroids Toxin that medicine, microorganism produce etc..
" double functional copolymer " refers to can be special with other parts (including but not limited to amino acid side group) comprising two The opposite sex reacts the polymer of the discontinuous functional group to form covalently or non-covalently key connection.Can be with particular organisms activity with one The functional group of radical reaction in component and another can connect with the difunctionality of the group of the radical reaction on the second biological components Connecing son can be used for formation to include the binding element of the first biological active component, bifunctional linker and the second biological active component. The known numerous programs and connection molecule for making various compounds be connected with peptide.For example, referring to European Patent Publication case the 188th, 256 Number, U.S. Patent No. 4,671,958, No. 4,659,839, No. 4,414,148, No. 4,699,784, the 4,680th, No. 338 and the 4th, 569, No. 789, it is to be incorporated herein by reference." multifunctional polymer " refer to comprising two or Two or more can be formed covalently or non-covalently with other parts (including but not limited to amino acid side group) specific reaction The polymer of bonded discontinuous functional group.Double functional copolymer or multifunctional polymer can have any required length or molecule Amount, and can be chosen to provide special between one or more molecules and its acceptor or FGF-21 being connected with FGF-21 Interval or conformation needed for fixed.
When the conventional chemical formulas by writing from left to right represents substituent, the structure by writing from right to left is likewise covered by Obtained chemically identical substituent, for example, structure-CH2O- is equal to structure-OCH2-。
Term " substituent " includes but is not limited to " noiseless substituent "." noiseless substituent " is the stable chemical combination of generation The group of thing.Suitable noiseless substituent or group include but is not limited to halogen, C1-C10Alkyl, C2-C10Alkenyl, C2-C10 Alkynyl, C1-C10Alkoxy, C1-C12Aralkyl, C1-C12Alkaryl, C3-C12Cycloalkyl, C3-C12Cycloalkenyl group, phenyl, it is substituted Phenyl, toluyl, xylyl, xenyl, C2-C12Alkoxyalkyl, C2-C12Alkoxy aryl, C7-C12Aryloxy group alkyl Base, C7-C12Epoxide aryl, C1-C6Alkyl sulphinyl, C1-C10Alkyl sulphonyl ,-(CH2)m-O-(C1-C10Alkyl) (wherein m For 1 to 8), aryl, substituted aryl, be substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic, 4-nitro alkyl ,- NO2、-CN、-NRC(O)-(C1-C10Alkyl) ,-C (O)-(C1-C10Alkyl), C2-C10Alkyl alkylthio base ,-C (O) O- (C1-C10Alkane Base) ,-OH ,-SO2,=S ,-COOH ,-NR2, carbonyl ,-C (O)-(C1-C10Alkyl)-CF3、-C(O)-CF3、-C(O)NR2、-(C1- C10Aryl)-S- (C6-C10Aryl) ,-C (O)-(C1-C10Aryl) ,-(CH2)m-O-(CH2)m-O-(C1-C10Alkyl) (wherein each m For 1 to 8) ,-C (O) NR2、-C(S)NR2、-SO2NR2、-NRC(O)NR2、-NRC(S)NR2, its salt etc..As used herein, each R For H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl or alkaryl.
Term " halogen " includes fluorine, chlorine, iodine and bromine.
Unless otherwise indicated, otherwise term " alkyl " itself or mean during the part as another substituent straight chain or Side chain or cyclic hydrocarbon group or its combination, its can for fully saturated, single insatiable hunger and/or how unsaturated and may include to have and specify Carbon atom number (i.e. C1-C10Mean 1 to 10 carbon) divalent group and multivalence group.The example of saturated hydrocarbyl include (but It is not limited to) following group, such as methyl, ethyl, n-propyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, hexamethylene Base, (cyclohexyl) methyl, Cvclopropvlmethvl；Homologue and isomery (such as) n-pentyl, n-hexyl, n-heptyl, n-octyl Body.Unsaturated alkyl is the alkyl with one or more double or triple bonds.The example of unsaturated alkyl includes (but not limiting In) vinyl, 2- acrylic, crotyl, 2- isopentene groups, 2- (butadienyl), 2,4- pentadienyls, 3- (Isosorbide-5-Nitraes-pentadiene Base), acetenyl, 1- propinyls and 3- propinyls, 3- butynyls and higher homologue and isomers.Unless otherwise indicated, it is no Then term " alkyl " is also intended to include the derivative of the alkyl defined in greater detail below, such as " miscellaneous alkyl ".It is only limited to alkyl Alkyl is referred to as " homology alkyl (homoalkyl) ".
The divalent group as derived from alkane is meant when term " alkylidene " itself or the part as another substituent, Such as (but not limited to) structure-CH2CH2- and-CH2CH2CH2CH2-, and further comprise being hereinafter described as " sub- miscellaneous alkyl " Group.Generally, alkyl (or alkylidene) will have 1 to 24 carbon atoms, wherein the base with 10 or less than 10 carbon atoms Group is the specific embodiment of method and composition specifically described herein." low-carbon alkyl " or " lower " is generally with 8 Individual or less than 8 carbon atoms relatively short-chain alkyl or alkylidene.
Term " alkoxy ", " alkyl amino " and " alkylthio group " (or thio alkoxy) is used with its conventional meaning, and And refer to the alkyl of remainder connection respectively by oxygen atom, amino or sulphur atom and molecule.
Unless otherwise indicated, otherwise term " miscellaneous alkyl " itself or mean when being combined with another term by specifying number Carbon atom and at least one be selected from the stable straight or branched that constitutes of hetero atom or ring-type by O, N, Si and S group constituted Alkyl or its combination, and wherein nitrogen and sulphur atom can optionally oxidized and nitrogen heteroatom can be optionally through quaternized.Miscellaneous original Sub- O, N and S and Si can be located at the position of any interior location of miscellaneous alkyl or the remainder connection positioned at alkyl and molecule. Example includes but is not limited to-CH2-CH2-O-CH3、-CH2-CH2-NH-CH3、-CH2-CH2-N(CH3)-CH3、-CH2-S-CH2- CH3、-CH2-CH2-S(O)-CH3、-CH2-CH2-S(O)2-CH3,-CH=CH-O-CH3、-Si(CH3)3、-CH2- CH=N-OCH3 With-CH=CH-N (CH3)-CH3.At most two hetero atoms can be adjacent, such as-CH2-NH-OCH3With-CH2-O-Si(CH3)3.Class As, mean the bilvalent radical as derived from miscellaneous alkyl when term " sub- miscellaneous alkyl " itself or the part as another substituent Group, such as (but not limited to)-CH2-CH2-S-CH2-CH2- and-CH2-S-CH2-CH2-NH-CH2-.For sub- miscellaneous alkyl, phase With or different hetero atoms can also occupy chain one or both ends (including but not limited to alkylidene epoxide, alkylenedioxy group, Alkylidene amino, alkylenediamino, aminooxy group alkylidene etc.).In addition, coming for alkylidene and sub- miscellaneous alkyl linking group Say, the direction that the chemical formula of linking group is write is not offered as the orientation of linking group.For example, formula-C (O)2R '-table Show-C (O)2R '-and-R ' C (O)2-。
Unless otherwise indicated, otherwise term " cycloalkyl " and " Heterocyclylalkyl " itself or time-division other table is combined with other terms Show the annular form of " alkyl " and " miscellaneous alkyl ".Therefore, to include saturation, part unsaturated and completely not for cycloalkyl or Heterocyclylalkyl The ring of saturation is bonded.In addition, for Heterocyclylalkyl, hetero atom can occupy the position of the remainder connection of heterocycle and molecule Put.The example of cycloalkyl includes but is not limited to cyclopenta, cyclohexyl, 1- cyclohexenyl groups, 3- cyclohexenyl groups, suberyl etc..It is miscellaneous The example of cycloalkyl include but is not limited to 1- (1,2,5,6- tetrahydro pyridyl), 1- piperidyls, 2- piperidyls, 3- piperidyls, 4- morpholinyls, morpholinyl, tetrahydrofuran -2- bases, tetrahydrofuran -3- bases, thiophane -2- bases, thiophane -3- bases, 1- Piperazinyl, 2- piperazinyls etc..In addition, the term covers bicyclic and tricyclic structure.Similarly, term " sub- Heterocyclylalkyl " itself Or as another substituent a part when mean the divalent group as derived from Heterocyclylalkyl, and term " cycloalkylidene " this The divalent group by cycloalkyl-derivatized is meant when body or the part as another substituent.
As used herein, term " water-soluble polymer " refers to dissolve in any polymer in aqueous solvent.It is water-soluble Property polymer and FGF-21 polypeptides connection can produce following change, it including but not limited to increases relative to unmodified form Plus it is or adjusted serum half-life or increase or adjusted treatment half-life period, adjusted immunogenicity, adjusted Physical association characteristic (such as aggregation and polymer formed), the acceptor changed combine, being combined with one or more kinds of of changing The combination of collocation thing, and the receptor dimerization or poly changed.Water-soluble polymer may have or may not have itself Bioactivity, and can be used as connection FGF-21 and other materials (including but not limited to one or more FGF-21 Polypeptide or one or more kinds of bioactive molecule) connexon.Suitable polymer include but is not limited to polyethylene glycol, Methoxy PEG-propionaldehyde, its single C1-C10Alkoxy or aryloxy derivatives (it is described in U.S. Patent No. 5,252,714, it is described Patent is to be incorporated herein by reference), mono methoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, poly- ammonia Base acid, divinyl ether maleic anhydride, N- (2- hydroxypropyls)-Methacrylamide, glucan, glucan derivative (including sulphur Sour glucan), polypropylene glycol, PPOX/ethylene oxide copolymer, polyoxyethylated polyols, heparin, heparin fragment, Polysaccharide, oligosaccharides, glycan, cellulose and cellulose derivative (including but not limited to methylcellulose and carboxymethyl cellulose), Starch and starch derivatives, polypeptide, poly- alkane glycol and its derivative, the copolymer of poly- alkane glycol and its derivative, polyvinyl Ether and alpha-beta-poly- [(2- hydroxyethyls)-DL- asparagines etc., or its mixture.The example bag of the water-soluble polymer Include (but not limited to) polyethylene glycol and seralbumin.
As used herein, term " poly- alkane glycol " or " poly- (alkane glycol) " refer to polyethylene glycol (PEG), gathered Propane diols, polytetramethylene glycol and its derivative.Term " poly- alkane glycol " cover linear and branched polymers and mean molecule quantity between Between 0.1kDa and 100kDa.Other exemplary embodiments are listed in (such as) commercial supplier catalogue, such as Shearwater Corporation catalogue " Polyethylene Glycol and Derivatives for BiomedicalApplications”(2001)。
Unless otherwise indicated, otherwise term " aryl " mean can for it is monocyclic or be fused together or be covalently attached it is polycyclic The how unsaturated aromatic hydrocarbon substituent of (including but not limited to 1 to 3 rings).Term " heteroaryl " refers to containing 1 to 4 choosings From N, O and S heteroatomic aryl (or ring), wherein nitrogen and sulphur atom is optionally oxidized, and nitrogen-atoms is optionally through season Ammonium.Heteroaryl can be connected by the remainder of hetero atom and molecule.The non-limiting examples of aryl and heteroaryl include benzene Base, 1- naphthyls, 2- naphthyls, 4- xenyls, 1- pyrrole radicals, 2- pyrrole radicals, 3- pyrrole radicals, 3- pyrazolyls, 2- imidazole radicals, 4- imidazoles Base, pyrazinyl, 2- oxazolyls, 4- oxazolyls, 2- phenyl -4- oxazolyls, 5- oxazolyls, 3- isoxazolyls, 4- isoxazolyls, 5- Isoxazolyl, 2- thiazolyls, 4- thiazolyls, 5- thiazolyls, 2- furyls, 3- furyls, 2- thienyls, 3- thienyls, 2- pyrroles Piperidinyl, 3- pyridine radicals, 4- pyridine radicals, 2- pyrimidine radicals, 4- pyrimidine radicals, 5- benzothiazolyls, purine radicals, 2- benzimidazolyls, 5- Indyl, 1- isoquinolyls, 5- isoquinolyls, 2- quinoxalinyls, 5- quinoxalinyls, 3- quinolyls and 6- quinolyls.Above-mentioned virtue The substituent of each of base and heteroaryl ring-member is both selected from the group of acceptable substituent described below.
For simplicity, when being applied in combination with other terms (including but not limited to aryloxy group, aryl sulphur epoxide, Aryl alkyl), term " aryl " includes aryl as defined above and heteroaryl ring.Therefore, term " aryl alkyl " is intended The group (including but not limited to benzyl, phenethyl, pyridylmethyl etc.) being connected including aryl with alkyl, the alkyl includes The alkyl of carbon atom (including but not limited to methylene) (for example) oxygen atom displacement (includes but is not limited to phenoxy group first Base, 2- pyridyloxymethyls, 3- (1- naphthyls epoxide) propyl group etc.).
Terms above (including but not limited to " alkyl ", " miscellaneous alkyl ", " aryl " and " heteroaryl ") each intends bag Include specified group is substituted and is unsubstituted form.The exemplary substituent of all types of groups provides as follows.
Alkyl and miscellaneous alkyl (including be commonly referred to as alkylidene, it is alkenyl, sub- miscellaneous alkyl, miscellaneous thiazolinyl, alkynyl, cycloalkyl, miscellaneous The group of cycloalkyl, cycloalkenyl group and heterocycloalkenyl) substituent can be in a variety of groups below (but not limited to) One or more kinds of groups：- OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R " ' ,-OC (O) R′、-C(O)R′、-CO2R′、-CONR′R″、-OC(O)NR′R″、-NR″C(O)R′、-NR′-C(O)NR″R″′、-NR″C(O)2R ' ,-NR-C (NR ' R " R " ')=NR " " ,-NR-C (NR ' R ")=NR " ' ,-S (O) R ', S (O)2R′、-S(O)2NR′R″、- NRSO2R ' ,-CN and-NO2, its number is in the range of 0 to (2m '+1), and wherein m ' is the sum of carbon atom in such group. R ', R ", R " ' and R " " refer to hydrogen, are substituted or are unsubstituted miscellaneous alkyl, are substituted or be unsubstituted aryl (its independently of one another Aryl including but not limited to through the substitution of 1-3 halogen), be substituted or be unsubstituted alkyl, alkoxy or thio alkoxy Or aryl alkyl.For example, when the compound of the present invention includes more than one R group, each R group is independently through choosing Select, and when there is one of R ', R ", R " ' and R " " group above, these groups are equally chosen independently of one another.When When R ' and R " are connected with same nitrogen-atoms, it can combine to form 5 yuan, 6 yuan or 7 yuan of rings with nitrogen-atoms.For example ,-NR ' R " are beaten Calculate and include but is not limited to 1- pyrrolidinyls and 4- morpholinyls.According to the above-mentioned discussion to substituent, the technology people of art Member should be appreciated that term " alkyl " is intended to include the group of the carbon atom combined with the group in addition to hydrogen-based, such as alkylhalide group (including but not limited to-CF3With-CH2CF3) and acyl group (including but not limited to-C (O) CH3、-C(O)CF3、-C(O) CH2OCH3Deng).
Similar with for the substituent described in alkyl, the substituent of aryl and heteroaryl, which exists, to be changed and selected from (but not limiting In)：Halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R " ' ,-OC (O) R ', C (O) R′、-CO2R′、-CONR′R″、-OC(O)NR′R″、-NR″C(O)R′、-NR′-C(O)NR″R″′、-NR″C(O)2R′、-NR-C (NR ' R " R " ')=NR " " ,-NR-C (NR ' R ")=NR " ' ,-S (O) R ' ,-S (O)2R′、-S(O)2NR′R″、-NRSO2R′、-CN With-NO2、-R′、-N3、-CH(Ph)2, fluorine (C1-C4) alkoxy and fluorine (C1-C4) alkyl, its number opens on 0 to aromatic ring system Put in the total scope of valence state；And wherein R ', R ", R " ' and R " " are independently selected from hydrogen, alkyl, miscellaneous alkyl, aryl and miscellaneous Aryl.For example, when the compound of the present invention includes more than one R group, each R group is independently chosen, and is worked as When there is one of R ', R ", R " ' and R " " group above, these groups are equally chosen independently of one another.
As used herein, term " adjusted serum half-life " means the circulating half-life phase of modified FGF-21 Positive change or negative change for its unmodified form.By the Each point in time blood sampling after administration FGF-21 and determine The concentration of molecule described in each sample measures serum half-life.The correlation of serum-concentration and time allow to calculate serum half Decline the phase.The increase of serum half-life is preferably at least about twice, but for example when smaller increase can facilitate satisfactorily to Prescription case or when avoiding toxic action, smaller increase is also applicable.In certain embodiments, the increase is at least about 3 times, extremely It is few about 5 times or at least about 10 times.
As used herein, term " adjusted treatment half-life period " means the FGF-21 of therapeutically effective amount half-life period Relative to the positive change or negative change of its unmodified form.The medicine for measuring molecule by the Each point in time after administration is moved Mechanics and/or drug effect property treat half-life period to measure.Increased treatment half-life period can ideally facilitate it is specific it is beneficial to Prescription case, specific beneficial accumulated dose avoid unwanted effect.In certain embodiments, it is increased treatment half-life period be by Effect increase, the combination through decorating molecule and its target are increased or decreased, enzyme (such as protease) increases the decomposition of molecule or Reduction or unmodified molecule another parameter or mechanism of action increase or decrease or molecule receptor-mediated removing increase or Reduction causes.
When applied to nucleic acid or protein, term " through separation " represents the nucleic acid or protein without it in natural shape At least some components in the cellular component associated under state, or the nucleic acid or protein be concentrated to higher than it is in vivo or living The external degree for producing concentration.It can be homogeneous state.It can be dry or partial desiccation state through separate substance, or be solution (bag Include the (but not limited to) aqueous solution) form.It can be include other pharmaceutically acceptable supporting agents and/or excipient medical group The component of compound.It is pure to determine usually using the technique of analytical chemistry such as polyacrylamide gel electrophoresis or high performance liquid chromatography Degree and homogenieity.It is substantially purified as the protein of main matter present in preparation.Specifically, through isolated genes It is the ORFs separation of the protein from gene described in side joint and coding in addition to gene of interest.Term " purified " table Show that nucleic acid or protein produce a substantial band in running gel.Specifically, its meaning is probably nucleic acid or albumen Matter is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure or purer.
Term " nucleic acid " refer to the deoxyribonucleotide of single-stranded or double-stranded form, dezyribonucleoside, ribonucleotide or Ribonucleotide and its polymer.Except nonspecific limitation, otherwise the term is covered containing similar known to natural nucleotide The nucleic acid of thing, the nucleic acid has the binding property similar to reference nucleic acid and is with the side similar to naturally occurring nucleotides Formula is metabolized.Unless in addition specific limitation, otherwise the term also refer to oligonucleotide analogs, it include PNA (peptide nucleic acid), instead The DNA analogs (thiophosphate, phosphoramidate etc.) used in adopted technology.Unless otherwise indicated, otherwise specific nucleic acid sequence Row also impliedly cover its conservative modification variant (including but not limited to degenerate codon substitution) and complementary series and bright The sequence really indicated.Specifically, can be by producing the 3rd position of (or all) codons selected by one or more Blended base and/or deoxyinosine residue substitution sequence come realize degenerate codon replace (Batzer et al., Nucleic Acid Res.19：5081(1991)；Ohtsuka et al., J.Biol.Chem.260：2605-2608(1985)；Rossolini Et al., Mol.Cell.Probes 8：91-98(1994)).
Term " polypeptide ", " peptide " and " protein " is used interchangeably herein to refer to the polymer of amino acid residue. That is, the description for polypeptide is equally applicable to the description to peptide and the description to protein, and vice versa.Institute It is non-naturally encoded amino that term, which is stated, suitable for naturally occurring amino acid polymer and one or more amino acid residues The amino acid polymer of acid.As used herein, the term covers the amino acid chain of any length, including full length protein, Wherein amino acid residue is connected by covalent peptide bonds.
Term " amino acid " refers to naturally occurring and non-naturally-occurring amino acid, and with naturally occurring amino acids seemingly The amino acid analogue that works of mode and amino acid simulant.Natural coded amino acid is 20 kinds of common amino acid (third ammonia Acid, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, Leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and Pyrrolysine and selenocysteine.Amino acid analogue refers to have to be tied with the basic chemistry of naturally occurring amino acid identical The compound of structure (that is, the α carbon combined with hydrogen, carboxyl, amino and R group), such as homoserine, nor-leucine, methionine are sub- Sulfone, methionine methyl sulfonium.The analog has through modifying R group (for example, nor-leucine) or through modifying peptide backbone, but still protects Hold and naturally occurring amino acid identical basic chemical structure.To referring to including for example naturally occurring protein sources for amino acid L-amino acid, D- amino acid；Amino acid through chemical modification, such as Amino acid variants and derivative；Naturally occurring non-egg White exogenous amino acid, such as Beta-alanine, ornithine；And with the property that amino acid characteristics are known as in art Chemical synthesis compound.The example of non-naturally-occurring amino acid includes but is not limited to Alpha-Methyl amino acid (for example, Alpha-Methyl third Propylhomoserin), D- amino acid, class histidine amino acid (for example, 2- amino-histidines, beta-hydroxy-histidine, high histidine, α- Methyl fluoride-histidine and Alpha-Methyl-histidine), there is in side chain the amino acid (" height " amino acid) of methylene, and side in addition The amino acid (for example, cysteine) that carboxylic acid functional in chain is replaced through sulfonic group.
Herein, amino acid can entrust by its commonly known three letter symbols or by IUPAC-IUB biological chemical names The one-letter symbol of member's meeting (IUPAC-IUB Biochemical Nomenclature Commission) recommendation is referred to.Together Sample, single letter code that nucleotides can generally be received by it is referred to.
" conservative sex modification variant " is applied to amino acid and nucleotide sequence.For specific nucleic acid sequence, " conservative Modification variant " refers to the nucleic acid for encoding consistent or substantially consistent amino acid sequence, or when nucleic acid not coded amino acid sequence Refer to substantially consistent sequence during row.Due to the degeneracy of genetic code, a large amount of function identical nucleic acid codings are any given Protein.For example, codon GCA, GCC, GCG and GCU all coded amino acid alanine.Therefore, in alanine by password At each position that son is specified, codon can change into any one in the corresponding codon without many coded by change Peptide.The variance is " silent variant (silent variation) ", and it is one kind in conservative sex modification variation.Herein Each nucleotide sequence of middle coded polypeptide also describes each possible silent variant of nucleic acid.Those skilled in the art should Recognize, (in addition to AUG and TGG, AUG is usually the unique codon of methionine to each codon in nucleic acid, and TGG is usual For the unique codon of tryptophan) function identical molecule can be all produced through modifying.Therefore, the nucleic acid of coded polypeptide is each heavy Silent variation is all lain in each sequence.
On amino acid sequence, those skilled in the art should be understood that in change, addition or the coded sequence of missing Single amino acid or a small amount of amino acid to indivedual substitutions of nucleic acid, peptide, polypeptide or protein sequence, lack or be added to " conservative sex modification variant ", wherein the change will cause amino acid deletions, amino acid addition or amino acid through chemically class As 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.The known conservative replaces table that the similar amino acid of function is provided of those skilled in the art.It is described to protect Sex modification variant in addition to for polymorphic variant (and being not excluded for these variants) is kept, is also inter-species homologue and the present invention Allele.
The known conservative replaces table that the similar Amino acid of function is provided of those skilled in the art.Below eight groups it is each self-contained There is the amino acid each other for conservative replaces：
1) alanine (A), glycine (G)；
2) aspartic acid (D), glutamic acid (E)；
3) asparagine (N), glutamine (Q)；
4) arginine (R), lysine (K)；
5) isoleucine (I), leucine (L), methionine (M), valine (V)；
6) phenylalanine (F), tyrosine (Y), tryptophan (W)；
7) serine (S), threonine (T)；With
8) cysteine (C), methionine (M)
(referring for example to Creighton, Proteins：Structures and Molecular Properties(W H Freeman&Co.；Second edition (in December, 1993)).
In the situation of two or more nucleic acid or peptide sequence, term " consistent " or " uniformity " percentage refer to Two or more sequences of identical or subsequence.When comparing by comparing window and comparing maximum correspondence, or such as use One kind in one sequence comparison algorithm (or the available other algorithms of those skilled in the art) or by manual alignment and Visually inspect and measure during designated area, if sequence has and identical (that is, has about 60% uniformity in designated area, about 65%th, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% uniformity) amino acid residue or nucleotides hundred Divide ratio, then the sequence is " substantially consistent ".This defines the complementary series for also referring to cycle tests.Uniformity may be present In on the region that length is at least about 50 amino acid or nucleotides, or the region that length is 75-100 amino acid or nucleotides On, or in the whole sequence of (when not specified) polynucleotides or polypeptide.It can be encoded by the method comprised the steps of The polynucleotides (including homologue from non-human species) of polypeptide of the present invention：Being used under stringent hybridization condition has the present invention Polynucleotide sequence or its fragment labeled probe screening library, and separation full-length cDNA and contain the polynucleotides The genomic clone of sequence.Those skilled in the art knows the hybridization technique.
For sequence comparatively, a usual sequence serves as the reference sequences compared with cycle tests.Using sequence During row comparison algorithm, cycle tests and reference sequences are inputted in computer, subsequence coordinates are specified if necessary, and specify sequence Row algorithm routine parameter.Default program parameters can be used, or may specify alternate parameter.Then, sequence comparison algorithm is according to program Parameter calculates percentage of sequence identity of the cycle tests relative to reference sequences.
As used herein, " comparing window " include with reference to being selected from by 20 to 600, normally about 50 to about 200, more typically from about The section of any one of the continuous position number of group of 100 to about 150 compositions, wherein can be by sequence and with same number Continuous position reference sequences optimal comparison after two sequences are compared.Those skilled in the art becomes known for ratio Compared with sequence alignment method.It can carry out comparing for the optimal sequence that compares by the following method, methods described includes (but not limiting In) Smith and Waterman (1970) Adv.Appl.Math.2：482c local homology algorithm；Needleman and Wunsch (1970)J.Mol.Biol.48：443 homology alignment algorithm；Search for Pearson and Lipman (1988) Proc.Nat ' l.Acad.Sci.USA 85：2444 similarity method；(Wisconsin Genetics are implemented in the computerization of the algorithm GAP in Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, BESTFIT, FASTA and TFASTA)；Or manual alignment and visual inspection are (referring for example to Ausubel et al., Current Protocols in Molecular Biology (nineteen ninety-five supplementary issue)).
Suitable for determine percentage of sequence identity and sequence similarity percentage algorithm an example for BLAST and The algorithms of BLAST 2.0, it is described in Altschul et al., (1997) Nuc.Acids Res.25：3389-3402 and Altschul et al., (1990) J.Mol.Biol.215：In 403-410.For carry out BLAST analyses software can by via American National Biotechnology Information center (the National Center that WWW is accessed on ncbi.nlm.nih.gov ForBiotechnology Information) open acquisition.BLAST algorithm parameter W, T and X by determine compare sensitivity and Speed.BLASTN programs (for nucleotide sequence) use word length (W) 11, desired value (E) 10, M=5, N=-4 and two Bar chain is worth more by default.For amino acid sequence, BLASTP programs using word length 3 and desired value (E) 10 and BLOSUM62 score matrixes are (referring to Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.USA 89：10915) Than being worth more by default for logarithm (B) 50, desired value (E) 10, M=5, N=-4 and two chains.Generally at " low-complexity " Filter carries out BLAST algorithm in the case of closing.
BLAST algorithm also carry out similitude between two sequences statistical analysis (referring for example to Karlin and Altschul(1993)Proc.Natl.Acad.Sci.USA 90：5873-5787).The similitude provided by BLAST algorithm One is measured as minimum sum probability (smallest sum probability, P (N)), and it is provided on two nucleotides or ammonia The index for the probability that can be accidentally matched between base acid sequence.For example, if in test nucleic acid and the ratio of reference nucleic acid Middle minimum sum probability is less than about 0.2 or less than about 0.01, or less than about 0.001, then think the nucleic acid and reference sequences It is similar.
Phrase " with ... selectivity (or specificity) hybridization " refer to when complex mixture (including but not limited to total cell or Library DNA or RNA) in when there is specific nucleotide sequence, under stringent hybridization condition molecule only combined with the sequence, duplex Or hybridization.
Phrase " stringent hybridization condition " refer to DNA under known low ionic strength and hot conditions in the art, The hybridization of RNA, PNA or other nucleic acid mimics or the sequence of its combination.Generally, under strict conditions, probe can be with it in core Target subsequence hybridization in the complex mixture (including but not limited to total cell or library DNA or RNA) of acid, but not with answering Other sequence hybridizations in hybrid compound.Stringent condition has sequence dependent and under various circumstances can be different.Longer sequence Arrange specific hybrid at relatively high temperatures.Tijssen, Laboratory are found in the extensive guidance that nucleic acid hybridizes Techniques in Biochemistry and Molecular Biology-Hybridization with NucleicProbes, " Overview of principles of hybridization and the strategy of In nucleic acid assays " (1993).It is than specificity under regulation ionic strength, pH value to be typically chosen stringent condition Thermal melting point (the T of sequencem) low about 5 DEG C -10 DEG C.TmIt is in for 50% and target sequences hybridization of the probe complementary with target Balance is (because target sequences are present in excess, in TmUnder, balance when occupy 50% probe) when temperature it is (strong in specified ion Under degree, pH value and nucleic acid concentration).Stringent condition can be pH value be 7.0 to 8.3 time salinity less than about 1.0M sodium ions, Normally about 0.01M to 1.0M Na ion concentrations (or other salt) and temperature are for short probe (including but not limited to 10 to 50 Nucleotides) for be at least about 30 DEG C and for long probe (including but not limited to more than 50 nucleotides) at least about 60 DEG C of condition.Also stringent condition can be realized by adding the destabilizing agent such as formamide.For selectivity or specificity For hybridization, positive signal can be at least twice background, optionally 10 times of background hybridizations.Exemplary stringent hybridization condition can be such as Under：50% formamide, 5 × SSC and 1%SDS, are cultivated at 42 DEG C；Or 5 × SSC, 1%SDS, cultivated at 65 DEG C, wherein Washed at 65 DEG C in 0.2 × SSC and 0.1%SDS.The washing can carry out 5,15,30,60,120 minutes or longer time.
As used herein, term " eucaryote " refers to belong to systematic growth domain (phylogenetic domain) very The organism of core field (Eucarya), such as animal (including but not limited to mammal, insect, reptile, birds), Infusorian, plant (including but not limited to monocotyledon, dicotyledon, algae etc.), fungi, yeast, flagellate, micro- spore Sub- worm, protist etc..
As used herein, term " non-eucaryote " refers to non-most eukaryotes.For example, non-most eukaryotes can Belong to eubacteria (Eubacteria) and (include but is not limited to Escherichia coli, thermus thermophilus (Thermusthermophilus), bacillus stearothermophilus (Bacillus stearothermophilus), fluorescence are false single Born of the same parents bacterium (Pseudomonas fluorescens), pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas putida Bacterium (Pseudomonas putida) etc.) systematic growth domain, or Archimycetes (Archaea) (including but not limited to Zhan Shi methane Coccus (Methanococcus jannaschii), thermophilic hot autotrophic methane bacteria (Methanobacteriumthermoautotrophicum), (the thermophilic salt of such as walsh is richly endowed for Halophiles (Halobacterium) Bacterium (Haloferaxvolcanii) and Halophiles kind NRC-1), it is the ancient bacterium (Archaeoglobus fulgidus) of hyperthermophilic, strong Red-hot coccus (Pyrococcus furiosus), the ancient bacterium (Pyrococcus horikoshii) of extreme thermophilic, the life of thermophilic spring are ancient Bacterium (Aeuropyrum pernix) etc.) systematic growth domain.
As used herein, term " individual " refers to animal, is in certain embodiments mammal, and in other realities It is the mankind to apply in example, and it is treatment, the object of observation or experiment.Animal can be companion animals (for example, dog, cat etc.), domestic animal (example Such as, ox, sheep, pig, horse etc.) or experimental animal (for example, rat, mouse, guinea pig etc.).
As used herein, term " effective dose " refers to the amount through modifying non-natural amino acid polypeptides of institute's administration, described Amount can mitigate one or more kinds of symptoms of treated disease, symptom or illness to a certain extent.Can administration contain this Described in text preventative, enhancement and/or therapeutic treatment are carried out through modifying the composition of non-natural amino acid polypeptides.
The effect of effect or duration needed for term " enhancing " means increase or extension.Therefore, treated for enhancing For the effect of agent, term " enhancing " refers to increase or extends other therapeutic agents to the effect of the effect of system or duration Ability.As used herein, " enhancing effective dose " refers to be enough to strengthen the amount of effect of another therapeutic agent in required system.When During in patient, on this purposes, effectively amount will be depending on following factor：The seriousness and disease of disease, illness or symptom Journey, previous therapies, the health status of patient and reaction and the judgement of attending doctor to medicine.
As used herein, term " through modification " refers to giving any change that polypeptide is made, for example polypeptide length, many Amino acid sequence, chemical constitution, common translation modification or the change of posttranslational modification of peptide.Form " (through modification) " term is meant The polypeptide discussed is optionally through modification, that is to say, that the polypeptide discussed can be through modification or unmodified.
Term " posttranslational modification " refers to the relatively described ammonia after natural or alpha-non-natural amino acid is had been incorporated into polypeptide chain Any modification that base acid occurs.Only for example, the term covers that common translation is in vivo modified, common translation is in vitro modified In vitro modified after in vivo modifying and translate after (such as in not celliferous translation system), translation.
In prophylactic applications, by the composition administration containing FGF-21 polypeptides be susceptible to suffer from specified disease, illness or symptom or Patient in the risk for suffering from specified disease, illness or symptom.The amount is defined as " prevention effective dose ".In this purposes In, depending on health status, the body weight of precise volume also regarding patient etc..Think by normal experiment (for example, dosage escalation clinic examination Test) determine the prevention effective dose completely in the range of the technical ability of art.
Term " through protection " refers to exist " the guarantor for preventing chemical reactivity functional group from being reacted under special reaction condition Protect base " or part.Protection group changes the type of the chemically reactive group depending on being protected.For example, if chemistry is anti- Answering property group is amine or hydrazides, then protection group may be selected from tertbutyloxycarbonyl (t-Boc) and 9- fluorenylmethoxycarbonyl groups (Fmoc) Group.If chemically reactive group is mercaptan, then protection group can be adjacent pyridyl disulfide.If chemical reactivity Group is carboxylic acid (such as butyric acid or propionic acid) or hydroxyl, then protection group can be for benzyl or such as methyl, ethyl or the tert-butyl group Alkyl.Known other protection groups can also be used in method and composition specifically described herein or and methods described in art It is used together with composition, it includes the photo-labile group such as Nvoc and MeNvoc.Known other guarantors in art Shield base can also be used for being used together with composition in method and composition specifically described herein or with methods described.
Only for example, end-blocking/blocking group may be selected from：
Other protection groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd edition, in John Wiley ＆ Sons, New York, NY, 1999, it is to be incorporated herein in entirety by reference.
In treatment use, it will be enough to cure or at least partly suppress disease, the amount of the symptom of illness or symptom contains The patient of the disease, symptom or illness is had been inflicted with through modifying the composition administration of non-natural amino acid polypeptides.The amount is fixed Justice is " therapeutically effective amount ", and will be depending on following factor：Disease, the seriousness and the course of disease of illness or symptom, previous therapies, trouble The health status of person and reaction and the judgement of attending doctor to medicine.Think by normal experiment (for example, dosage escalation faces Bed experiment) determine the therapeutically effective amount completely in the range of the technical ability of art.
Term " treatment " is to be used to refer to preventative and/or therapeutic treatment.
Non-naturally encoded amino acids polypeptide presented herein may include one or more atoms through atomic mass or The chemical combination through isotope marks of the mass number atomic substitutions different from atomic mass typically seen in nature or mass number Thing.The example for the isotope that may be incorporated into the compounds of this invention is included exemplified by the isotope of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, difference Such as2H、3H、13C、14C、15N、18O、17O、35S、8F、36Cl.It is specifically described herein it is some through isotope marks compound (for example, It is associated with for example3H and14The radioisotopic compound such as C) it can be used in medicine and/or substrate tissue distribution calibrating.This Outside, with such as deuterium (i.e.2The isotope substitution such as H) can provide some treatment advantages for thanking to stability generation by higher generation, for example, increase Plus vivo half-life or reduction volume requirements.
All isomers (including but not limited to diastereoisomer, enantiomter and its mixture) are considered as this A part for composition described in text.In other or other embodiments, non-naturally encoded amino acids polypeptide to need production It is metabolized after the organism administration of raw metabolin, the metabolin is used subsequently to produce required effect, and (including required treatment is made With).In other or another embodiment, it is the active metabolite of non-naturally encoded amino acids polypeptide.
In some cases, non-naturally encoded amino acids polypeptide can exist with tautomeric forms.In addition, institute herein The non-naturally encoded amino acids polypeptide stated can in unsolvated form and with pharmaceutically acceptable solvent (such as water, second Alcohol etc.) formed solvation form exist.It is also considered as the solvation form disclosed herein.Those skilled in the art should Recognize, some compounds herein can exist with some tautomeric forms.All this tautomeric forms all by regarding Make a part for composition specifically described herein.
Unless otherwise indicated, otherwise using the mass spectrum in the range of the technical ability of art, NMR, HPLC, protein chemistry, Biochemistry, recombinant DNA technology and pharmacological conventional method.
Embodiment
I. introduction
The present invention provides the FGF-21 molecules for including at least one alpha-non-natural amino acid.In certain embodiments of the present invention In, the FGF-21 polypeptides with least one alpha-non-natural amino acid include at least one posttranslational modification.In one embodiment, At least one described posttranslational modification, which is included, utilizes the known change for being applied to specific reactivity group of those skilled in the art Method, makes the molecule comprising the second reactive group connect with least one alpha-non-natural amino acid comprising the first reactive group Connect, the molecule for including the second reactive group includes but is not limited to：Mark, dyestuff, polymer, water-soluble polymer, Derivative, photocrosslinking agent, radionuclide, cytotoxic compound, medicine, affinity marker, the photoaffinity of polyethylene glycol Mark, reactive compounds, resin, the second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal-chelating Agent, co-factor, aliphatic acid, carbohydrate, polynucleotides, DNA, RNA, antisense polynucleotides, carbohydrate, water-soluble dendroid are gathered Compound, cyclodextrin, inhibition ribonucleic acid, biomaterial, nano-particle, spin labeling, fluorogen, the part containing metal, put Penetrating property part, novel functional groups, group, light cage part, actinic radiation with other molecule covalents or noncovalent interaction The part that can excite, can photoisomerization part, biotin, biotin derivative, biotin analog, be combined with heavy atom Partly, the group that chemically cracks, can photodestruciton group, the side chain of extension, sugar, redox active agent, the ammonia of carbon connection Base thio-acid, toxin part, the part through isotope marks, biophysics probe, phosphorescence groups, chemiluminescent groups, electronics Dense group, magnetic group, insertion group, chromophore, energy transfer agent, bioactivator, detectable label, small molecule, amount Sub- point, nanometer transmission element, radioactive nucleotides, radioactivity are transmitted any combinations of element, neutron capture agent or above-mentioned substance, or appointed Compound or material what needed for it.For example, the first reactive group is alkynyl moiety (including but not limited in non-day Right amino acid is in propargyloxyphenylalanine, wherein propargyl is sometimes referred to as acetylene moiety), and the second reactivity Group is azide moiety, and utilizes [3+2] cycloaddition chemical method.In another example, the first reactive group is nitrine Partly (including but not limited in alpha-non-natural amino acid in azido-L-phenylalanine), and the second reactive group is Alkynyl moiety.In some embodiments of the modified FGF-21 polypeptides of the present invention, turned over using at least one comprising at least one The alpha-non-natural amino acid (including but not limited to the alpha-non-natural amino acid containing keto functional group) modified after translating, wherein it is described extremely A few posttranslational modification includes sugar moieties.In certain embodiments, posttranslational modification is in eukaryotic or non-eukaryotic In in vivo carry out.The molecule can be connected by connexon, polymer, water-soluble polymer or other molecules with polypeptide.Molecule It can be directly connected to polypeptide.
In certain embodiments, protein is repaiied after including the translation that at least one is in vivo carried out by a kind of host cell Decorations, wherein the posttranslational modification can not generally be carried out by another host cell species.In certain embodiments, protein Including the posttranslational modification that at least one is in vivo carried out by eukaryotic, wherein the posttranslational modification can not generally pass through Non- eukaryotic is carried out.The example of posttranslational modification includes but is not limited to glycosylation, acetylation, acylation, lipid-modified, palm Acylation, palmitate addition (palmitate addition), phosphorylation, glycolipids key modification etc..In one embodiment, translate Modification includes and makes oligosaccharides be connected with asparagine by GlcNAc- asparagine keys (to include but is not limited to wherein oligosaccharides bag afterwards Containing (GlcNAc-Man)2- Man-GlcNAc-GlcNAc etc.).In another embodiment, posttranslational modification is included and passed through GalNAc- serines, GalNAc- threonines, GlcNAc- serines or GlcNAc- threonine keys make oligosaccharides (including (but not limit In) Gal-GalNAc, Gal-GlcNAc etc.) be connected with serine or threonine.In certain embodiments, protein of the invention Or polypeptide can include secretion or positioning sequence, epitope label, FLAG labels, polyhistidyl tags, GST fusions etc.. The example of secretory signal sequence includes but is not limited to prokaryotes secretory signal sequence, eucaryote secretory signal sequence, is Carry out bacterial expression and optimize 5 ' eucaryote secretory signal sequence, novel secretory signal sequence, pectin lyase enzyme secretion letter Number sequence, OmpA secretory signal sequences and bacteriophage secretory signal sequence.The example of secretory signal sequence includes but is not limited to STII (prokaryotes), Fd GIII and M13 (bacteriophage), Bgl2 (yeast) and the signal sequence bla from transposon.It is any Such sequence all can be through modifying so that polypeptide has required result, including but not limited to one letter of unlike signal sequence substitution Number sequence, replaces targeting sequencing etc. with different targeting sequencings.
Protein of interest or polypeptide can containing at least one, at least two, at least three, at least four, at least five It is individual, at least six, at least seven, at least eight, at least nine or ten or more than ten alpha-non-natural amino acids.The non-natural Amino acid may be the same or different, for example, may be present 1 in protein, more than 2,3,4,5,6,7,8,9,10 or 10 different positions Point, it is comprising 1, more than 2,3,4,5,6,7,8,9,10 or 10 different alpha-non-natural amino acids.In certain embodiments, albumen At least one (but all or less than) specific amino acids in the presence of the naturally occurring form of matter are replaced through alpha-non-natural amino acid.
The present invention provides the method and composition based on the FGF-21 comprising at least one non-naturally encoded amino acids.It is near Few non-naturally encoded amino acids is introduced into FGF-21 can allow using be related to specified chemical reaction (include but is not limited to One or more non-naturally encoded amino acids react without 20 kinds of amino acid reactions with generally existing) engagement chemistry. In certain embodiments, the FGF-21 comprising non-naturally encoded amino acids be by the side chains of non-naturally encoded amino acids with it is water-soluble Property polymer (such as polyethylene glycol (PEG)) connection.The present invention provides a kind of height of use PEG derivatives selectively modifying proteins Efficacious prescriptions method, its be related to response selection codon by non-genetic coding amino acid (including but not limited to containing 20 kinds it is natural simultaneously Enter the amino acid of undiscovered functional group or substituent (including but not limited to ketone, nitrine or acetylene moiety) in amino acid) choosing Selecting property is incorporated in protein, and then with suitable reactivity amino acid described in PEG Derivatives Modifieds.Once it is incorporated to amino acid Side chain, so that it may by using known to those skilled in the art be applied to non-naturally encoded amino acids present in particular functional The chemical method of group or substituent carrys out modified amino acid side chain.A variety of known chemical methodes are applied in the present invention will be water-soluble Property polymer is incorporated in protein.Methods described include but is not limited to respectively with (including but is not limited to) acetylene or Azide Thing derivative carries out Hughes's root [3+2] cycloaddition reaction (referring for example to Padwa, A.Comprehensive Organic Synthesis, volume 4, (1991) Trost, B.M. volumes, Pergamon, Oxford, the 1069-1109 pages；And Huisgen, R.1,3-Dipolar CycloadditionChemistry, (1984) Padwa, A. volumes, Wiley, New York, 1-176 Page).
Because Hughes's root [3+2] cycloaddition method is related to cycloaddition rather than nucleophilic substitution, protein can be in pole Through modification under high selectivity., can at room temperature under aqueous conditions by adding Cu (I) salt of catalytic amount into reactant mixture This reaction is carried out with quality region selectivity (1,4 ＞ 1,5).For example, referring to Tornoe et al., (2002)J.Org.Chem.67： 3057-3064；With Rostovtsev et al., (2002)Angew.Chem.Int.Ed.41：2596-2599；And WO03/ 101972.The molecule that can be added to by [3+2] cycloaddition on present protein includes with suitable functional group or taken Dai Ji (including but not limited to azido or acetylene-derivative) substantially any molecule.These molecules can be added to respectively to be had The alpha-non-natural amino acid (including but not limited to propargyloxyphenylalanine) of acetenyl or the non-natural with azido On amino acid (including but not limited to azido-phenylalanine).
5 yuan of rings obtained by Hughes's root [3+2] cycloaddition are usually irreversible in reproducibility environment, and aqueous For a long time to hydrolysis-stable in environment.Therefore, active PEG Derivatives Modifieds that can be with the present invention under harsh aqueous conditions are a variety of The Physical and chemical characteristics of material.Even more importantly, because azide moiety and acetylene moiety have each other specificity (and For example not with the reaction of any of 20 kinds of common genetic coding amino acids), so can be in one or more specificity With high selective modification protein in site.
The present invention also provides the water solubility and hydrolysis-stable derivative of PEG derivatives and with one or more second The related hydrophilic polymer of alkynes or azide moiety.PEG polymer derivants containing acetylene moiety are with high selectivity and Respond selection codon and be selectively introduced the coupling of the azide moiety in protein.Similarly, the PEG containing azide moiety gathers Compound derivative is selectively introduced the coupling of the acetylene moiety in protein with high selectivity with having responded selection codon.
More particularly, azide moiety is including (but not limited to) alkyl azide, aromatic yl azide and these nitrine The derivative of compound.The derivative of alkyl and aromatic yl azide may include other substituents, as long as keeping acetylene specificity anti- Answering property.Acetylene moiety includes alkyl and aryl ethane and respective derivative.The derivative of alkyl and aryl ethane can be wrapped Other substituents are included, as long as keeping azide specific reaction.
The present invention provide have a variety of functional groups, substituent or partial material and the binding element of other materials, it is described its Its material includes but is not limited to mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent； Radionuclide；Cytotoxic compound；Medicine；Affinity marker；Photoaffinity is marked；Reactive compounds；Resin；Second Protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate； Polynucleotides；DNA；RNA；Antisense polynucleotides；Carbohydrate；Water-soluble dendritic；Cyclodextrin；Inhibition ribonucleic acid； Biomaterial；Nano-particle；Spin labeling；Fluorogen；Part containing metal；Radioactive segment；Novel functional groups；With other points The group that son covalently or non-covalently interacts；Light cage part；The part that actinic radiation can be excited；Can photoisomerization portion Point；Biotin；Biotin derivative；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；Can light The group of cracking；The side chain of extension；The sugar of carbon connection；Redox active agent；Aminothio acid；Toxin part；Through isotope The part of mark；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；Insert group； Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Quantum dot；Nanometer transmission element；Radioactivity nucleosides Acid；Radioactivity transmission element；Neutron capture agent；Or any combinations of above-mentioned substance, or any other required compound or material.This Invention also includes the material with nitrine or acetylene moiety and the PEG polymer derivants with corresponding acetylene or azide moiety Binding element.For example, the PEG polymer containing azide moiety can contain the non-heredity with acetylene functional groups in protein It is coupled at the position of coded amino acid with bioactive molecule.Make that PEG and bioactive molecule be coupled is bonded including (but not limiting In) Hughes's root [3+2] cycloaddition product.
Clearly determined in art, PEG can be used for modified biological material surface (referring for example to United States Patent (USP) 6, 610,281；Mehvar, R., J.Pharm Pharm Sci., 3 (1)：125-136 (2000), it is to be herein incorporated by reference Herein).The present invention also includes the biology for including the surface with one or more reactive azido or acetylene sites Material, and nitrine is contained by the bonded one or more present invention being coupled with the surface of Hughes's root [3+2] cycloaddition The polymer of base or acetylene.Biomaterial and other materials also can be by other bonded in addition to azido or acetylene are bonded (such as by including the bonded of carboxylic acid, amine, alcohol or thiol moiety) and the polymer derivant activated by azide or acetylene Coupling is used for subsequent reactions to leave nitrine or acetylene moiety.
The present invention includes a kind of method containing azido polymer and containing acetylene polymer for synthesizing the present invention.Containing nitrine In the case of the PEG derivatives of base, the carbon atom of azide and polymer can be made directly to be bonded.Or, can be by by one end Bridging agent with azide moiety is connected to prepare the PEG derivatives containing azido with Conventional activation polymer so that gained gathers Compound has azide moiety in its end.In the case of the PEG derivatives containing acetylene, the carbon atom of acetylene and polymer can be made Directly it is bonded.Or, it can be prepared by there is one end the bridging agent of acetylene moiety be connected with Conventional activation polymer containing second The PEG derivatives of alkynes so that resulting polymers have acetylene moiety in its end.
More particularly, in the case of the PEG derivatives containing azido, the water with least one activity hydroxy part The reaction of soluble polymer experience has reactive stronger part (such as methanesulfonate, trifluoro ethyl sulfonic acid root, toluene sulphur to produce Acid group or halogen leaving group) be substituted polymer.Those skilled in the art it is known containing sulfonic acid halide, halogen atom and The preparation of the PEG derivatives of other leaving groups and use.The polymer that gained is substituted is then subjected to reaction with polymer The end stronger part of azide moiety substitution reaction.Or, the water with least one active nucleophilic or electrophilic subdivision The bridging agent that soluble polymer has azido with one end reacts so that formed altogether between PEG polymer and bridging agent Valence link, and azide moiety is located at the end of polymer.Nucleophilic known to those skilled in the art and electrophilic subdivision, it is wrapped Include amine, mercaptan, hydrazides, hydrazine, alcohol, carboxylate, aldehyde, ketone, thioesters etc..
More particularly, in the case of the PEG derivatives containing acetylene, with the water-soluble of at least one activity hydroxy part Property the reaction of polymer experience with from the precursor displacement halogen or other activated leaving groups containing acetylene moiety.Or, have The bridging agent that at least one active nucleophilic or the water-soluble polymer of electrophilic subdivision have acetylene with one end reacts so that Covalent bond is formed between PEG polymer and bridging agent, and acetylene moiety is located at the end of polymer.The technology of art Personnel are able adequately determines halogen moiety, the use of activated leaving group, nucleophilic and electrophilic subdivision in organic synthesis situation with And PEG derivatives preparation and use.
The present invention also provides a kind of selective modification protein other materials are added to through the side on modifying protein Method, the other materials include but is not limited to water-soluble polymer, and such as PEG and the PEG containing nitrine or acetylene moiety spread out It is biological.PEG derivatives containing azido and containing acetylene can be used for changing property (the wherein biocompatibility, steady of surface and molecule Qualitative, dissolubility and shortage immunogenicity are more important), and a kind of selectivity more previously known than in art is provided simultaneously Stronger makes the method that PEG derivatives are connected with protein.
II. fibroblast growth factor
Because FGF is for promoting growth, propagation, survival and the differentiation of various kinds of cell and organization type to have effective active, It is persistently used as the therapeutic agent of a variety of different indications, and the indication includes wound healing, such as muscle skeleton symptom, example Such as fracture, ligament and tissue repair, tendonitis, bursal synovitis；Skin symptom, such as burn, incised wound, breach, bedsore, ulcer healing It is slow etc.；For organization protection, repair, and induction of vascular is generated between miocardial infarction and local ischemic stage；For treating god Through symptom, such as neurodegenerative disorders and apoplexy；For treating eye disease, including macular degeneration etc..
Fibroblast growth factor (FGF) albumen differentiated so far belongs to the growth of regulation various kinds of cell type and divided The signal transducers family of change.FGF albumen for anthropophysiology and pathological importance to a certain extent with its Embry ogenesis, vascular development are relevant with the key effect in growth and bone growth.Ex vivo experiment has proven to FGF in regulation Work in the cell growth and division of endothelial cell, vascular smooth muscle cells, fibroblast and heart and Skeletal Muscle Cell With.Other members of FGF families and its biological agent are to be described in Crossley et al., Development, 121：439-451 (1995)；Ohuchi et al., Development, 124：2235-2244(1997)；Gemel et al., Genomics, 35：253- 257(1996)；And Ghosh et al., Cell Growth and Differentiation, 7：In 1425-1434 (1996).
FGF albumen is also most important to human health and disease because of the effect in growth of cancer cells.For example, FGF-8 through differentiating for induced in breast cancer and prostate gland cancer cell by androgen growth factor (Tanaka et al., FEBSLett.363：226-230 (1995) and P.N.A.S.89：8928-8932(1992)).
Effects of the FGF in normal development is illustrated to a certain extent by studying FGF receptor.Wilke, T. et al., Dev.Dynam.210：41-52 (1997) has found that FGFR1, FGFR2 and FGFR3 transcript are positioned at during the embryonic development of chicken In the specific region of head.In Ke Lusong syndromes (Crouzon syndrome) (bon e formation symptom in a kind of abnormal film), Expression pattern is relevant with the region influenceed by mankind's FGFR mutation.Belluardo, N. et al., Jour.Comp.Neur.379： 226-246 (1997) studies positioning of the FGFR 1,2 and 3mRNA in rat brain, and it was found that the cell in multiple brain area domains is special The opposite sex.In addition, FGFR1 and FGFR2mRNA is expressed in the reactive cell of astroglia after cerebral lesion, so as to support specific Effects of the FGF in cerebral disease and loss.Ozawa, K. et al., Mol.BrainRes.41：279-288 (1996) report births FGF1 and FGF-5 expression afterwards increased, and FGF3, FGF-6, FGF-7 and FGF-8 gene are shown in the expression ratio of late embryo stage The stage is high after birth.Co-factor Klotho beta (Klb) may also be relevant with the signal transduction of FGF-21 and its acceptor.Report Lead the ability that Klb increases FGFR1 and FGFR4 is combined with FGF21.Klb is unidirectional transmembrane protein, although and having no knowledge about completely The effect of cross-film form, but on FGF23 it has been shown that Klotho can strengthen FGF23 combines and increase the phosphorylation of FGF receptor, And Klotho beta shown enhancing FGF-21 combine (H.Kurosu, Y.Ogawa, M.Miyoshi, M.Yamamoto, A.Nandi, K.P.Rosenblatt, M.G.Baum, S.Schiavi, M.-C.Hu, O.W.Moe, M.Kuro-o, Regulation Offibroblast growth factor-23signaling by Klotho.J.Biol.Chem.281,6120-6123 (2006), it is incorporated herein by reference).
Katoh et al. (International Journal of Oncology (2006) 29：FGF family 163-168) is described Race and the phylogenetic analysis of family member.Katoh et al. also discusses the Signal transduction pathway network in intestines and stomach.
Plotnikov et al. (Cell (1999) 98：641-650) FGF2 of the description with FGF receptor 1 (FGFR1) crystalline substance Body structure and twice of the symmetrical dimer formed by two such compounds.Plotnikov et al. provide receptor dimerization and The model of dimerization is induced by FGF and heparin.
Future is it may be found that other members of FGF families.Can be by predicting computer assisted two grades of protein sequence Analyzed with tertiary structure and by differentiating FGF families designed for differentiating the selection technique of the molecule combined with specific target Newcomer.
Therefore, the description of FGF families is only in order at illustrative purpose and provided by way of example, and is not intended to limit this The scope of method, composition, strategy and technology described in text.This is led in addition, referring to that FGF-21 is intended in present application Claim the example of any member as FGF families.It is therefore to be understood that herein in connection with repairing described in FGF-21 polypeptides or protein Decorations and chemical reaction are equally applicable to any member of FGF families, including the specific member listed herein.
III. the general recombinant nucleic acid method used in the present invention
In numerous embodiments of the present invention, it will be separated, cloned using recombination method and generally change coding is of interest The nucleic acid of FGF-21 polypeptides.The embodiment is for (including but is not limited to) protein expression or for from FGF-21 polypeptides Variant, derivative, during the generation of expression cassette or other sequences.In certain embodiments, the sequence of polypeptide of the present invention is encoded Row are operably connected on allogeneic promoter.
Can be with the amino acid sequence of parent polypeptide (including but not limited to SEQ ID NO：Amino shown in 1-7 Acid sequence) based on carry out the nucleotide sequence of FGF-21 polypeptide of the composite coding comprising non-naturally encoded amino acids, and then Change the nucleotide sequence and (that is, lack or take to realize the introducing (that is, be incorporated to or replace) of relevant amino acid residue or remove Generation).Can be according to conventional methods by direct mutagenesis come easily modified nucleotide sequence.Or, it can be prepared by chemical synthesis Nucleotide sequence, will including but not limited to produce the place of recombinant polypeptide by using oligonucleotide synthesis device and prioritizing selection Preferred codon prepares nucleotide sequence in chief cell, and wherein oligonucleotides is the amino acid sequence according to required polypeptide It is designed.For example, it can be reacted by PCR, connection or link-chain type come the part of polypeptide needed for synthesizing and assembling coding Some small oligonucleotides.For example, referring to Barany et al., Proc.Natl.Acad.Sci.88：189-193(1991)；The U.S. Patent 6,521,427, it is to be incorporated herein by reference.
The present invention utilizes the routine techniques in genetic recombination field.Disclose the basis of the conventional method used in the present invention Article includes Sambrook et al., Molecular Cloning, A Laboratory Manual (the 3rd edition, 2001)； Kriegler, Gene Transfer and Expression：A Laboratory Manual(1990)；And Current Protocols inMolecular Biology (Ausubel et al. is compiled, 1994).
Describing the general article of molecular biotechnology includes Berger and Kimmel,Guide to Molecular CloningTechniques, Methods in Enzymology, volume 152,Academic Press, Inc., San Diego, CA (Berger)；Sambrook et al.,Molecular Cloning-A Laboratory Manual (second edition), The 1-3 volumes, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") andCurrent Protocols in Molecular Biology, F.M.Ausubel et al. compiles, Current Protocols, GreenePublishing Associates, Inc. and John Wiley ＆ Sons, Inc.'s Joint venture, (annual supplement in 1999) (" Ausubel ").These articles describe mutagenesis, carrier and promoter use and Many other related subjects, the related subject be related to and (include but is not limited to) including be used for prepare include alpha-non-natural amino acid, Orthogonal tRNA, orthogonal synthesis enzyme and its to protein selection codon gene or polynucleotides generation.
The present invention uses various types of mutagenesis for many purposes, and the purpose includes but is not limited to produce novel conjunction Into enzyme or tRNA, make tRNA molecular mutations, be mutated the polynucleotides of coding synzyme, produce tRNA libraries, produce synzyme Library, generation select codon, the selection codon for encoding alpha-non-natural amino acid are inserted in protein or polypeptide of interest. The mutagenesis include but is not limited to direct mutagenesis, random point mutagenesis, homologous recombination, DNA reorganization or other recurrence method of mutagenesis, Chimeric construct, the mutagenesis using the template containing uracil, oligonucleotides directed mutagenesis, the DNA mutagenesis of phosphorothioate, Mutagenesis using notched duplex DNA etc., the mutagenesis of PCT mediations, or its any combinations.Other suitable methods include point Mispairing reparation, the mutagenesis using reparation deficiency host strain, limitation select and limited purifying, deletion mutagenesis, pass through total gene Synthesize the mutagenesis carried out, double-strand break reparation etc..The mutagenesis that (including but is not limited to) is related to chimeric construct is also included within this hair In bright.In one embodiment, (it can be wrapped by the Given information of naturally occurring molecule or the naturally occurring molecule for having changed or being mutated Include (but not limited to) sequence, sequence compare, physical property, two grades, three or four structure, crystal structure etc.) instruct mutagenesis.
Visible article and example herein describe these programs.Other information is found in following discloses case and wherein quoted Bibliography in：Ling et al., Approaches to DNA mutagenesis：Ab overview,Anal Biochem.254(2)：157-178(1997)；Dale et al., Oligonucleotide-directed random Mutagenesis using thephosphorothioate method,Methods Mol.Biol.57：369-374 (1996)；Smith, In vitromutagenesis,Ann.Rev.Genet.19：423-462(1985)；Botstein and Shortle, Strategies andapplications of in vitro mutagenesis,Science229：1193- 1201(1985)；Carter, Site-directedmutagenesis,Biochem.J.237：1-7(1986)；Kunkel, The Efficiency of oligonucleotide directedmutagenesis,Nucleic Acids & Molecular Biology(Eckstein, F. and Lilley, D.M.J are compiled, SpringerVerlag, Berlin) (1987)；Kunkel, Rapid And efficient site-specific mutagenesis-withoutphenotypic selection,Proc.Natl.Acad.Sci.USA82：488-492(1985)；Kunkel et al., Rapid andefficient site- Specific mutagenesis without phenotypic selection,Methods in Enzymol.154,367- 382(1987)；Bass et al., Mutant Trp repressors with new DNA-binding specificities,Science242：240-245(1988)；Zoller and Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors：an efficient and general procedure for the Production of pointmutations in any DNA fragment,Nucleic Acids Res.10：6487- 6500(1982)；Zoller and Smith, Oligonucleotide-directed mutagenesis of DNA Fragments cloned into M13 vectors,Methodsin Enzymol.100：468-500(1983)；Zoller And Smith, Oligonucleotide-directed mutagenesis：asimple method using two Oligonucleotide primers and a single-stranded DNA template,Methods in Enzymol.154：329-350(1987)；Taylor et al., The use ofphosphorothioate-modified DNA In restriction enzyme reactions to prepare nicked DNA,Nucl.Acids Res.13：8749- 8764(1985)；Taylor et al., The rapid generation ofoligonucleotide-directed Mutations at high frequency using phosphorothioate-modified DNA,Nucl.Acids Res.13：8765-8785(1985)；Nakamaye and Eckstein, Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and its Application tooligonucleotide-directed mutagenesis,Nucl.Acids Res.14：9679- 9698(1986)；Sayers et al., 5 ' -3 ' Exonucleases in phosphorothioate-based Oligonucleotide-directed mutagenesis,Nucl.Acids Res.16：791-802(1988)；Sayers etc. People, Strand specific cleavage of phosphorothioate-containing DNA by reaction With restriction endonucleases in the presenceofethidium bromide, (1988)Nucl.Acids Res.16：803-814；Kramer et al., The gapped duplexDNA approach to Oligonucleotide-directed mutationconstruction,Nucl.Acids Res.12：9441-9456 (1984)；Kramer and Fritz Oligonucleotide-directed construction of mutations Viagapped duplex DNA,Methods in Enzymol.154：350-367(1987)；Kramer et al., Improvedenzymatic in vitro reactions in the gapped duplex DNA approach to Oligonucleotide-directedconstruction of mutations,Nucl.Acids Res.16：7207 (1988)；Fritz et al., Oligonucleotide-directed construction of mutations：a gapped Duplex DNA procedure withoutenzymatic reactions in vitro,Nucl.Acids Res.16： 6987-6999(1988)；Kramer et al., Difierentbase/base mismatches are corrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E.coli,Cell38：879-887(1984)；Carter et al., Improvedoligonucleotide site-directed Mutagenesis using Ml3 vectors,Nucl.Acids Res.13：4431-4443(1985)；Carter, Improved oligonucleotide-directed mutagenesis using Ml3 vectors,Methodsin Enzymol.154：382-403(1987)；Eghtedarzadeh and Henikoff, Use of oligonucleotides Togenerate large deletions,Nucl.Acids Res.14：5115(1986)；Wells et al., Importance Ofhydrogen-bond formation in stabilizing the transition state of subtilisin,Phil.Trans.R.Soc.Lond.A 317：415-423(1986)；Nambiar et al., Total synthesis and Cloning of a gene codingfor the ribonuclease S protein,Science223：1299-1301 (1984)；Sakamar and Khorana, Totalsynthesis and expression of a gene for the alpha-subunit of bovine rod outer segment guaninenucleotide-binding protein (transducin),Nucl.Acids Res.14：6361-6372(1988)；Wells et al., Cassette mutagenesis：an efficient method for generation of multiple mutations at Definedsites,Gene34：315-323(1985)；Grundstrom et al., Oligonucleotide-directed Mutagenesis bymicroscale ' shot-gun ' gene synthesis,Nucl.Acids Res.13：3305-3316 (1985)；Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli：Amethod for site-specific mutagenesis,Proc.Natl.Acad.Sci.USA, 83：7177-7181(1986)；Arnold, Protein engineering for Unusual environments,Current Opinion in Biotechnology4：450-455(1993)；Sieber etc. People, Nature Biotechnology, 19：456-460(2001)；W.P.C.Stemmer,Nature370,389-91 (1994)；And I.A.Lorimer, I.Pastan,Nucleic Acids Res.23,3067-8 (1995).On numerous The other details for stating method are found inMethods in EnzymologyIn volume 154, the document also describes to lure for various The effective control for the trouble shooter problem that change method is brought.
Generally according to by Beaucage and Caruthers, Tetrahedron Letts.22 (20)：1859-1862, (1981) the solid phase phosphoramidite triester method described by, such as using such as Needham-VanDevanter et al., Nucleic Acids Res., 12：Automated synthesizer described in 6159-6168 (1984) is chemically synthesized for example for the present invention's Oligonucleotides in mutagenesis (for example, making synzyme library be mutated or change tRNA).
The present invention also relates to by orthogonal tRNA/RS to be in vivo incorporated to alpha-non-natural amino acid eukaryotic host cell, Non- eukaryotic host cell and organism.With the present invention polynucleotides or including polynucleotides of the present invention construct (including (but Be not limited to) present invention carrier, it can be such as cloning vector or expression vector) with genetic terms it is engineered (including (but It is not limited to) conversion, transduction or transfection) host cell.For example, orthogonal tRNA, orthogonal tRNA/synthetase and the derivative egg of desire The code area of white matter is operably connected on the gene expression control elements worked in required host cell.Carrier can be Such as plasmid, clay (cosmid), bacteriophage, bacterium, virus, naked polynucleotides or the form for engaging polynucleotides.Pass through mark Carrier is introduced into cell and/or microorganism by quasi- method, methods described include electroporation (Fromm et al.,Proc.Natl.Acad.Sci.USA82,5824 (1985)), by viral vector infection, in beads or particle Medium Culture or On the surface by with nucleic acid small particles high velocity ballistic infiltration (Klein et al.,Nature327,70-73 (1987)) etc..
Can be in the improved conventional nutrient culture for being suitable to the activity such as screening step, activating promoters or selection transformant The host cell that culturing engineering was transformed in base.These cells can be optionally through cultivating and entering in transgenic organism.(including But it is not limited to) include on other useful bibliography of Cell isolation and culture (for example, on follow-up nucleic acid separate) Freshney(1994)Culture of Animal Cells, a Manual of Basic Technique, the 3rd edition, Wiley-Liss, New York and references cited therein；Payne et al., (1992)Plant Cell and Tissue Culturein Liquid SystemsJohn Wiley ＆ Sons, Inc.New York, NY；Gamborg and Phillips (eds.) (1995)Plant Cell, Tissue and Organ Culture；Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook ofMicrobiological Media(1993) CRC Press, Boca Raton, FL.
Several well known methods that labeled target nucleic acid is introduced into cell can be used, any of which method can be used in this hair In bright.These methods include：Recipient cell and the bacterial protoplast fusion containing DNA, electroporation, particle bombardment (projectile bombardment) and through viral vector (being discussed further below) infection etc..Bacterial cell can use In the number of the plasmid of DNA construct of the amplification containing the present invention.Make bacterial growth to exponential phase and can be by affiliated Plasmid in field in known a variety of method separation of bacterial (referring for example to Sambrook).In addition, commercially available kit with from Plasmid purification is (referring for example to the EasyPrep from Pharmacia Biotech in bacteriumTM、FlexiPrepTM；Come from Stratagene StrataCleanTM；And the QIAprep from QiagenTM).Then, further manipulate and separate and purify The plasmid crossed carrys out transfectional cell using the plasmid or is incorporated into relevant carriers with infection biological to produce other plasmids Body.Typical carriers contain transcription and translation terminator, transcription and translation homing sequence and available for adjusting specific labeled target nucleic acid Expression promoter.Carrier optionally includes universal expression box, and it contains at least one independent terminator sequence, allows institute State sequence (including but not limited to shuttle vector) and use that expression cassette is replicated in eukaryotic or prokaryotic or both In the selected marker of prokaryotic system and eukaryotic system.Carrier is suitable to replicate and integrate in prokaryotic, eukaryotic or both. Referring to Giliman and Smith,Gene8：81(1979)；Roberts et al.,Nature.328：731(1987)； Schneider, B. et al.,Protein Expr.Purif.6435：10(1995)；Ausubel, Sambrook, Berger are ( With above).The catalogue of bacterium and bacteriophage suitable for clone is provided by (such as) ATCC, for example, published by ATCCThe ATCC Catalogue of Bacteria and Bacteriophage(1992), Gherna et al. (eds.).For being sequenced, gram The other base programs and basic theory Consideration of the other side of grand and molecular biology are also seen in Watson et al. (1992)Recombinant DNA second editionsIn ScientificAmerican Books, NY.In addition, substantially any nucleic acid (and actually any labeled nucleic acid, standard or non-standard) all can from any of a variety of commercial sources customization or Standard is ordered, and (Midland, TX can pass through these commercial sources such as Midland Certified Reagent Company WWW existsmcrc.comIt is upper obtain), The Great American Gene Company (Ramona, CA, can pass through ten thousand dimension Net existsgenco.comIt is upper obtain), (Chicago, IL's ExpressGen Inc. can be existed by WWWexpressgen.com It is upper to obtain), OperonTechnologies Inc. (Alameda, CA) and many other sources.
Select codon
The selection codon of the present invention extends the genetic code subframe of Protein synthesis mechanism.For example, select Select codon and include but is not limited to unique three base codon, nonsense codon (such as terminator codon, including (but do not limit In) amber codon (UAG), ochre codon or opal codon (UGA)), unnatural codons, four or more Codon, rare codon of base etc..Those skilled in the art is it is clear that gene or polynucleotides needed for can introducing In selection codon number range it is very wide, its including but not limited to coding FGF-21 polypeptides at least one of list Exist in one polynucleotides one or more, two or more, three or more than three, 4,5,6,7, 8,9,10 or 10 codons selected above.
In one embodiment, methods described is directed to use with as the selection codon of terminator codon with vivo simultaneously Enter one or more alpha-non-natural amino acids.For example, identification terminator codon (including but not limited to UAG) is produced O-tRNA, and make O-tRNA aminoacylated using required alpha-non-natural amino acid by O-RS.This O-tRNA can not be by naturally depositing Recognized in the aminoacyl-tRNA synthetase of host.Routine direct mutagenesis can be used at the site of interest in polypeptide of interest Introduce terminator codon (including but not limited to TAG).For example, referring to Sayers, J.R. et al., (1988), 5 ' -3 ' Exonuclease inphosphorothioate-based oligonucleotide-directed mutagenesis.Nucleic Acids Res, 16：791-802.As O-RS, O-tRNA and the nucleic acid of coding polypeptide of interest When in vivo combining, respond UAG codons and be incorporated to alpha-non-natural amino acid to obtain containing non-natural ammonia in specified location The polypeptide of base acid.
In vivo being incorporated to alpha-non-natural amino acid can be carried out in the case where not interfering significantly with eukaryotic host cell.Citing comes Say because for UAG codons suppression efficiency depend on O-tRNA (including but not limited to amber suppressor tRNA) with Eucaryon releasing factor (including but not limited to eRF) (it is combined and originated the peptide that ribosomes release is growing with terminator codon) Between competition, so the expression that can increase O-tRNA and/or inhibiting factor tRNA by (including but is not limited to) be adjusted Section suppresses efficiency.
Rare codon can also be used to encode alpha-non-natural amino acid.For example, when in vitro protein synthetic reaction In arginine concentrations reduction when, it has therefore proved that rare arginine codon AGG is effective for the synthesis by being acylated through alanine TRNA inserts Ala.For example, referring to Ma et al.,Biochemistry, 32：7939(1993).In this case, tRNA is synthesized With the naturally occurring tRNA Arg competitions existed in Escherichia coli as minor materials.Some organisms are without using all three Body codon.Utilize micrococcus luteus (Micrococcus luteus) in not specified codon AGA in vitro transcribe/ Amino acid is inserted in translation extract.For example, referring to Kowal and Oliver,Nucl.Acid.Res.,25：4685(1997).Can The component of the present invention is produced in vivo to use these rare codons.
Select codon also comprising extension codon, it includes but is not limited to the password of four or more base Son, the codon of such as four, five, six or more than six bases.The example of four base codons includes but is not limited to AGGA, CUAG, UAGA, CCCU etc..The example of five base codons include but is not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC etc..The feature of the present invention based on frameshit including the use of being suppressed (frameshift suppression) Extension codon.The codon of four or more base can be by (including but is not limited to) one or more non-natural aminos In acid insertion same protein.For example, exist with anticodon loop (for example, with least 8-10nt anticode Subring) mutation O-tRNA (including but not limited to specific frameshift suppressor tRNA) in the case of, by four or four with The codon of upper base pronounces single amino acid.In other embodiments, anticodon loop decodable code (including but is not limited to) is extremely The password of few four base codons, at least one five base codon or at least one six base codon or more base Son.Because in the presence of 256 kinds of possible four base codons, the codon of four or more base can be used same Multiple alpha-non-natural amino acids are encoded in cell.Referring to Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size,Chemistry and Biology, 9：237-244；Magliery, (2001) Expanding the Genetic Code：Selectionof Efficient Suppressors of Four-base Codons and Identification of″Shifty″Four-baseCodons with a Library Approach In Escherichia coli,J.Mol.Biol.307：755-769.
For example, using in vitro biological synthesis method, alpha-non-natural amino acid is incorporated to four base codons In protein.For example, referring to Ma et al., (1993)Biochemistry, 32：7939；And Hohsaka et al., (1999)J.Am.Chem.Soc.121：34.Using CGGG and AGGU, thus in vitro using the frameshit of two chemical acylations suppress because The NBD derivatives of 2- naphthylalanines and lysine are incorporated in streptavidin by sub- tRNA simultaneously.For example, referring to Hohsaka et al., (1999)J.Am.Chem.Soc, 121：12194.In in vivo studying, Moore et al. researchs have The tRNALeu derivatives of NCUA anticodons suppress the ability of UAGN codons (N can be U, A, G or C), and it was found that tetrad UAGA can be decoded by the tRNALeu with UCUA anticodons with 13% to 26% efficiency, wherein few in 0 or -1 framework Decoding.Referring to Moore et al., (2000)J.Mol.Biol.,298：195.In one embodiment, it can use in the present invention Extension codon based on rare codon or nonsense codon, its missense that can reduce at other undesirable sites is led to Read and frameshit suppresses.
For given system, selection codon may also comprise natural three base codon, wherein endogenous system Without using (or being rarely employed) natural base codon.For example, this includes lacking natural three base codon of identification TRNA system and/or the system that three base codons are rare codon.
Codon is selected optionally to include nonnatural base pair.These nonnatural bases are to further expanding existing hereditary generation Code.One extra base-pair makes the number of triplet codon increase to 125 from 64.The property of 3rd base-pair includes stable And selective base pairing, by polymerase, with hi-fi, effectively enzymatic is incorporated in DNA and new in synthesis Nonnatural base lasting primer extend effective to after.May be adapted to the description of the nonnatural base pair of method and composition includes (for example) Hirao et al., (2002) An unnatural base pair for incorporating amino Acidanalogues into protein,Nature Biotechnology, 20：177-182.Referring also to Wu, Y. et al., (2002)J.Am.Chem.Soc.124：14626-14630.Other related publication are listed below.
For in vivo use, non-natural nucleoside is that film is permeable and phosphorylated to form corresponding three phosphorus Hydrochlorate.In addition, increased hereditary information is stable and is not destroyed by cellular enzymes.Work utilization previous Benner et al. and specification Watson-Crick matches different hydrogen bond syntypes, wherein most noticeable example is iso-C：Iso-G is matched.For example, Referring to Switzer et al., (1989)J.Am.Chem.Soc111：8322；And Piccirilli et al., (1990)Nature, 343：33；Kool, (2000)Curr.Opin.Chem.Biol.,4：602.These bases generally to a certain extent with trona Base mispairing and be unable to enzymatic duplication.Kool and colleague confirm that the hydrophobicity accumulation interaction between base can replace hydrogen bonding Drive the formation of base-pair.Referring to Kool, (2000)Curr.Opin.Chem.Biol.,4：602；And Guckian and Kool, (1998)Angew.Chem.Int.Ed.Engl., 36,2825.It is being directed to developing the non-natural alkali for meeting all above-mentioned requirements During base pair, Schultz, Romesberg and colleague systematically synthesize and have studied a series of unnatural hydrophobic alkali Base.It was found that PICS：PICS self pairs are more stable than natural base-pair, and can be by e. coli dna polymerase I Ke Lienuo pieces Section (Klenow fragment, KF) is effectively incorporated into DNA.For example, referring to McMinn et al., (1999)J.Am.Chem.Soc,121：11585-6；And Ogawa et al., (2000)J.Am.Chem.Soc, 122：3274.KF can be passed through To be enough to be used in the efficiency and selectivity synthesis 3MN of biological function：3MN self pairs.For example, referring to Ogawa et al., (2000)J.Am.Chem.Soc,122：8803.However, both bases all serve as the chain terminator for further replicating.Mutant DNA Polymerase has been developed recently, and it can be used for replicating PICS self pairs.In addition, may be duplicated for 7AI self pairs.For example, Referring to Tae et al., (2001)J.Am.Chem.Soc, 123：7439.Also novel metal base-pair Dipic has been developed：Py, its Form stable pairing afterwards with reference to Cu (II).Referring to Meggers et al., (2000)J.Am.Chem.Soc,122：10714.Because prolonging Long codon and unnatural codons are inherently orthogonal with native codon, institute with the inventive method can using this property come Orthogonal tRNA is produced so that it is used.
Can also be used translation bypath system (translational bypassing system) by alpha-non-natural amino acid simultaneously In polypeptide needed for entering.In translation bypath system, big sequence is incorporated in gene, but be not transcribed into protein.It is described Sequence, which contains, serves as the structure that induction ribosomes skips the sequence and restarts the prompting of translation in insertion downstream.
In certain embodiments, the present invention method and/or composition in protein of interest or polypeptide (or its Part) it is to be encoded by nucleic acid.Generally, nucleic acid includes at least one selection codon, at least two selection codons, at least three Select codon, at least four selection codons, at least five selection codons, at least six selection codons, at least seven Select codon, at least eight selection codons, at least nine selection codons, ten or ten codons selected above.
It can be used those skilled in the art known and method being described herein carry out mutagenesis albumen of interest The encoding gene of matter or polypeptide, so that it includes (such as) one or more selection codons to be incorporated to non-natural amino Acid.For example, the nucleic acid of mutagenesis protein of interest is so that it includes one or more selection codons, so as to provide One or more alpha-non-natural amino acids are incorporated to.The present invention includes appointing (for example) including at least one alpha-non-natural amino acid Any this variant (including but is not limited to mutant) form of what albumen.Similarly, the present invention also includes corresponding nucleic, That is, any nucleic acid with one or more selection codons for encoding one or more alpha-non-natural amino acids.
The nucleic acid molecules of coding protein of interest (such as FGF-21 polypeptides) can be easily made to be mutated with appointing in polypeptide What position at is introduced cysteine needed for.Cysteine is widely used in reactive molecule, water-soluble polymer, protein or many Other molecules are planted to introduce on protein of interest.The known institute for being suitable to being incorporated to cysteine into polypeptide of those skilled in the art The method in position, such as U.S. Patent No. 6 are needed, described in 608, No. 183 (it is incorporated herein by reference) Method, and Standard mutagenesis techniques.
IV. non-naturally encoded amino acids
A variety of non-naturally encoded amino acids are suitable for the present invention.Any number of non-naturally encoded amino acids can be introduced In FGF-21 polypeptides.In general, the substantial genetic coding amino acid common to 20 kinds of the non-naturally encoded amino acids of introducing (that is, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, different Leucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and figured silk fabrics ammonia Acid) it is in chemical inertness.In certain embodiments, non-naturally encoded amino acids include and non-existent official in 20 kinds of common amino acids Can group (including but not limited to azido, ketone group, aldehyde radical and aminooxy group) effectively and selectively stable engagement of reaction formation The side chain functionalities of thing., including the FGF-21 polypeptides of the non-naturally encoded amino acids containing azido functional group can for example The second polypeptides reactive with polymer (including but not limited to polyethylene glycol) or containing alkynyl moiety, to be formed due to azido React to form stable binding element caused by Hughes's root [3+2] cycloaddition product with alkynes functional group selectivity.
The general structure of a-amino acid is described as follows (Formulas I)：
Non-naturally encoded amino acids are usually that (wherein R group is except for 20 kinds of natural ammonia with structural formula listed above Any substituent outside substituent in base acid) any structure, and be applicable in the present invention.Because the present invention's is non- Natural coded amino acid is generally different only about side-chain structure and natural amino acid presence, thus non-naturally encoded amino acids with Amido link identical mode is formed in naturally occurring polypeptide (including but not limited to natural or non-naturally encoded with other amino acid Amino acid) form amido link.However, non-naturally encoded amino acids have the side-chain radical for differentiating it from natural amino acid.Lift Example for, R optionally comprising alkyl-, aryl-, acyl group-, ketone group-, azido-, hydroxyl-, hydrazine, cyano group-, halogen-, hydrazides, Alkenyl, alkynyl, ether, mercaptan, seleno-, sulfonyl-, boric acid ester group (borate), boric acid ester group (boronate), phosphate, phosphine Acyl group, phosphine, heterocyclic radical, ketenes, imines, aldehyde, ester, thio-acid, azanol, amino etc. or its any combinations.Of interest is applicable Other non-naturally-occurring amino acid in the present invention include but is not limited to comprising can the amino acid of photoactivated cross-linking agent, spin The amino acid of mark, Fluorescent amino acid, the amino acid with reference to metal, the amino acid containing metal, radioactive amino acids, with novelty The amino acid of functional group, the amino acid with other molecule covalents or noncovalent interaction, light cage and/or can photoisomerization The amino acid, amino acid comprising biotin or biotin analog, glycosylated amino acid (such as sugar substitution serine), other The amino acid of carbohydrate modification, the amino acid of ketone group containing, the amino acid comprising polyethylene glycol or polyethers, replace through heavy atom Amino acid, chemically cracking and/or can photodestruciton amino acid, with natural amino acid compared with have extend side chain (including (but Be not limited to) polyethers or long chain hydrocarbons, including but not limited to more than about 5 or more than about 10 carbon) amino acid, carbon containing connection Sugar amino acid, redox active amino acids, containing aminothio acid amino acid and include one or more toxicity Partial amino acid.
It is applicable to the exemplary non-naturally encoded amino acids in the present invention and suitable for being reacted with water-soluble polymer Including but not limited to there is the non-natural of carbonyl, aminooxy group, hydrazine, hydrazides, semicarbazides, azide and alkyne reaction group Coded amino acid.In certain embodiments, non-naturally encoded amino acids include sugar moieties.The example of the amino acid includes N- second Acyl group-L- glucose amido-Serine, N- acetyl group-L- galactolipins amido-Serine, N- acetyl group-L- glucose amido- L-threonine, N- acetyl group-L- glucose amido-altheine and O- mannoses amido-Serine.The reality of these amino acid Example also include naturally occurring N- between amino acid and sugar or O- it is bonded be there is usually no in nature covalent bond (including (but not limited to) alkene, oxime, thioether, acid amides etc.) displacement example.The example of these amino acid also includes naturally occurring protein In the sugar that there is usually no, such as 2-DG, 2- deoxy-galactoses.
A variety of non-naturally encoded amino acids provided herein be purchased from (such as) Sigma-Aldrich (St.Louis, MO, USA), Novabiochem (EMD Biosciences branch company, Darmstadt, Germany) or Peptech (Burlington, MA, USA).Not commercially available non-naturally encoded amino acids are optionally to synthesize as provided herein, or Synthesized using standard method known to those skilled in the art.On organic synthesis technology, referring for example to Fessendon and Fessendon,Organic Chemistry, (1982, second edition, Willard Grant Press, BostonMass.)； March,Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York)；And Carey and Sundberg,Advanced Organic Chemistry(the 3rd edition, A and part B, 1990, PlenumPress, New York).Referring also to U.S. Patent No. 7,045, No. 337 and the 7th, 083, No. 970, it is to be hereby incorporated herein by In.In addition to the alpha-non-natural amino acid containing novel side chain, the alpha-non-natural amino acid being applicable in the present invention is also optionally wrapped Containing the backbone structure through modification, the including but not limited to structure as illustrated by Formula II and III structure：
Wherein Z generally comprises OH, NH2, SH, NH-R ' or S-R '；X and Y may be the same or different, and generally comprise S or O；And R and R ' is optionally identical or different, is generally selected from the composition above for the R group described in the alpha-non-natural amino acid with Formulas I Partial identical inventory and hydrogen.For example, alpha-non-natural amino acid of the invention is optionally included as illustrated by Formula II and III Amino or carboxyl in substitution.Such alpha-non-natural amino acid include but is not limited to 'alpha '-hydroxy acids, α-thio-acid, α- Aminothio carboxylate, including but not limited to the side chain corresponding to 20 kinds of common natural amino acids or non natural side chain Alpha-non-natural amino acid.In addition, the substitution on α-carbon optionally includes but is not limited to L, D or α-α-disubstituted amino acid, example Such as D-Glu, D-alanine, D- methyl-O-tyrosines, aminobutyric acid.Other structures substitute includes cyclic amino acid, For example proline analogs and 3,4,6,7,8 and 9 yuan of membered ring proline analogues；β and γ amino acid, for example, be substituted β-the third ammonia Acid and γ-aminobutyric acid.
A variety of alpha-non-natural amino acids are based on the natural amino acid such as tyrosine, glutamine, phenylalanine and fitted For in the present invention.Tyrosine Analogues include but is not limited to contraposition substitution tyrosine, ortho position substitution tyrosine and The tyrosine of meta substitution, wherein be substituted tyrosine comprising (including but is not limited to) ketone group (including but not limited to acetyl group), Benzoyl, amino, hydrazine, azanol, mercapto, carboxyl, isopropyl, methyl, C6-C20Straight or branched hydrocarbon, saturation or unsaturation Hydrocarbon, O- methyl, polyether group, nitro, alkynyl etc..In addition, being also covered by polysubstituted aromatic ring.It is applicable to the glutamy in the present invention Amine analog includes but is not limited to Alpha-hydroxy derivative, γ-substitutive derivative, cyclic derivatives and the paddy ammonia of acid amides substitution Amide derivatives.The exemplary phenylalanine analogues being applicable in the present invention include but is not limited to the phenylpropyl alcohol of contraposition substitution Propylhomoserin, ortho position substitution phenylalanine and meta substitution phenylalanine, wherein substituent comprising (including but is not limited to) hydroxyl, Methoxyl group, methyl, pi-allyl, aldehyde, azido, iodo, bromo, ketone group (including but is not limited to acetyl group), benzoyl, alkynyl Deng.The particular instance for the alpha-non-natural amino acid being applicable in the present invention including but not limited to acetyl group-L-phenylalanine, O- methyl-L-tyrosines, L-3- (2- naphthyls) alanine, 3- methylphenylalanines, O-4- pi-allyls-TYR, 4- third Base-TYR, three-O- acetyl group-GlcNAc β-serine, L-3,4 dihydroxyphenylalanine (L-Dopa), fluorinated phenylalanine, isopropyl-L- Phenylalanine, to azido-L-phenylalanine, to acyl group-L-phenylalanine, to benzoyl-L-phenylalanine, L- phosphoric acid silk Propylhomoserin, phosphono serine, phosphono tyrosine, to iodophenylalanine, to bromophenyl alanine, to amino-L-phenylalanine, different Propyl group-L-phenylalanine and to propargyloxy-phenylalanine etc..It is applicable to a variety of alpha-non-natural amino acids in the present invention The example of structure be provided in (such as) entitled " In vivo incorporation of unnatural amino In acids " WO2002/085923.On other methionine analogs, referring also to Kiick et al., (2002) Incorporation of azidesinto recombinant proteins for chemoselective Modification by the Staudinger ligation,PNAS99：19-24, it is to be hereby incorporated herein by In.Entitled " Compositions Containing, MethodsInvolving, and Uses of Non-natural Amino Acids and Polypeptides " international application case the PCT/US06/47822nd (its by reference simultaneously Enter herein) standard reductive alkylation of aromatic amine moiety (including but not limited to p-Aminophenylalanine) is described, and reduce Property amination.
There is provided including alpha-non-natural amino acid (such as to (propargyloxy)-phenylalanine) in one embodiment The composition of FGF-21 polypeptides.Also provide include to (propargyloxy)-phenylalanine and (including but is not limited to) protein with/ Or the various compositions of cell.On the one hand, including to the composition of (propargyloxy)-phenylalanine alpha-non-natural amino acid enter one Step includes orthogonal tRNA.Alpha-non-natural amino acid can with orthogonal tRNA bonds (including but not limited to covalently bonded), it include (but It is not limited to) by aminoacyl key and orthogonal tRNA covalently bondeds, the 3 ' OH with orthogonal tRNA terminal ribose sugar or 2 ' OH covalent bonds Knot etc..
The chemical part that can be incorporated to by means of alpha-non-natural amino acid in protein provides a variety of advantages and the manipulation of protein Property.For example, unique reactivity of keto functional group allows using a variety of any one of hydrazine or reagents containing azanol of containing in work External and in vivo selective modification protein.For example, heavy atom alpha-non-natural amino acid is applicable to determine phase x-ray structure number According to.The locus specificity for carrying out heavy atom using alpha-non-natural amino acid introduce also for selection heavy atom position provides it is selective with Flexibility.For example, photoreactivity alpha-non-natural amino acid is (including but not limited to benzophenone and aromatic yl azide The amino acid of (include but is not limited to triazobenzene) side chain) allow in vivo and in vitro effective photo-crosslinking of protein.Light is anti- The example of answering property alpha-non-natural amino acid is including but not limited to azido-phenylalanine and to benzoyl-phenylalanine. Then, can by excite photoreactive group (offer time control) make the protein with photoreactivity alpha-non-natural amino acid with Meaning crosslinking.In an example, (include but is not limited to) in the case where using nuclear magnetic resonance and vibrational spectrum, can be by conduct (including but is not limited to) methyl through isotope marks of partial structurtes and dynamic (dynamical) probe replaces the first of alpha-non-natural amino acid Base.For example, alkynyl or azido functional group allow to pass through [3+2] cycloaddition reaction selective modification albumen using molecule Matter.
Be incorporated to the alpha-non-natural amino acid of the amino terminal of polypeptide can by R group (its be except in 20 kinds of natural amino acids Substituent outside any substituent) and NH different from generally existing in a-amino acid (referring to Formulas I)2The second of group is anti- Answering property group is constituted.Similar alpha-non-natural amino acid can be in carboxyl terminal be incorporated to different from a-amino acid (referring to Formulas I) generally Second reactive group of the COOH group of presence.
It may be selected or the alpha-non-natural amino acid of the design present invention is unavailable other in 20 kinds of natural amino acids to provide Characteristic.For example, it optionally can design or select alpha-non-natural amino acid to improve (such as) and have the alpha-non-natural amino acid Protein biological property.For example, can be optionally following to improve by including alpha-non-natural amino acid in protein Property：Toxicity, bio distribution (biodistribution), dissolubility, stability are (for example, heat endurance, hydrolytic stability, oxygen Change stability, enzymatic degradation resistance etc.), purifying and processing convenience, structural property, spectral quality, chemistry and/or photochemistry property The ability that matter, catalytic activity, oxidation-reduction potential, half-life period and other molecules (such as) are covalently or non-covalently reacted.
The structure of alpha-non-natural amino acid and synthesis：Carbonyl, carbonyl sample group, masked carbonyl, through protecting carbonyl and azanol Base
In certain embodiments, the present invention provides through the FGF-21 that oxime key is connected with the water-soluble polymer such as PEG.
Polytype non-naturally encoded amino acids are applied to form oxime key.These amino acid include but is not limited to containing The non-naturally encoded amino acids of carbonyl, dicarbapentaborane or azanol base.This amino acid is to be described in U.S. Patent Publication case the 2006/th No. 0194256, No. 2006/0217532 and No. 2006/0217289 and entitled " Compositionscontaining, methods involving, and uses of non-natural amino acids In and polypeptides " WO2006/069246, the document is to be incorporated herein in entirety by reference.Non- day Right coded amino acid is also described in U.S. Patent No. 7,083,970 and U.S. Patent No. 7,045,337, and the document is It is incorporated herein in entirety by reference.
Some embodiments of the present invention are utilized to pass through to acetyl phenyl alanine amino acid at one or more positions Substituted FGF-21 polypeptides.Synthesis to acetyl group-(+/-)-phenylalanine and an acetyl group-(+/-)-phenylalanine is described in Zhang, Z. et al., Biochemistry 42：In 6735-6746 (2003), the document is to be hereby incorporated herein by In.Other amino acid containing carbonyl or containing dicarbapentaborane can be similarly prepared in those skilled in the art.In addition, being wrapped herein The alpha-non-natural amino acid included it is non-limiting it is exemplary synthesize be presented in U.S. Patent No. 7,083,970 Fig. 4,24-34 and In 36-39, the patent is to be incorporated herein in entirety by reference.
Amino acid with electrophilic reactivity group allows a variety of reactions by nucleophilic addition connection molecule.Institute State electrophilic reactivity group including carbonyl (including ketone group and dicarbapentaborane), carbonyl sample group (its have be similar to carbonyl (including Ketone group and dicarbapentaborane) reactivity and it is similar with carbonyl in structure), (it can be easily converted to carbonyl to masked carbonyl (including ketone group and dicarbapentaborane)), or (it has similar with carbonyl (including ketone group and dicarbapentaborane) after deprotection through protecting carbonyl Reactivity).The amino acid includes the amino acid of the structure with formula (IV)：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
B is optional, and is the connexon selected from the group being made up of following group when it is present：Lower, Be substituted lower, low-carbon alkenylene, be substituted the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon, be substituted the sub- miscellaneous alkyl of low-carbon ,- O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein k is 1,2 Or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,- C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylidene)-,-C (O) N (R ')-,- CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene is substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、-N(R′)C(O)N(R′)-、-N(R′) C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N =,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each independently H, alkyl or substituted alkyl；J is
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R " is each independently H, alkyl, substituted alkyl or protection group, or when in the presence of more than one R " during group, two R " optionally forms Heterocyclylalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
R3And R4It is each independently H, halogen, low-carbon alkyl or is substituted low-carbon alkyl, or R3And R4Or two R3Group Optionally form cycloalkyl or Heterocyclylalkyl；
Or-A-B-J-R groups collectively form comprising at least one carbonyl (including dicarbapentaborane), through protect carbonyl (including Through protect dicarbapentaborane) or masked carbonyl (including masked dicarbapentaborane) bicyclic or tricyclic naphthenes base or Heterocyclylalkyl；
Or-J-R groups are collectively formed comprising at least one carbonyl (including dicarbapentaborane), through protecting carbonyl (including through protecting Protect dicarbapentaborane) or masked carbonyl (including masked dicarbapentaborane) monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl；
Condition is when A is phenylene and R3During respectively H, B is present；And when A is-(CH2)4- and R3Respectively H When, B is not -NHC (O) (CH2CH2)-；And when A and B is not present and R3During respectively H, R is not methyl.
In addition, including the amino acid of the structure with formula (V)：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon alkylene Base, lower, low-carbon alkenylene are substituted, the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon is substituted, is substituted the sub- miscellaneous alkane of low-carbon Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein K be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
Condition is that B is present when A is phenylene；And when A is-(CH2)4- when, B is not -NHC (O) (CH2CH2)-；And And when A and B are not present, R is not methyl.
In addition, including the amino acid of the structure with formula (VI)：
Wherein：
B is the connexon selected from the group being made up of following group：Lower, be substituted lower, it is low Carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon, is substituted the sub- miscellaneous alkyl of low-carbon ,-O- ,-O- (alkylidene or warp Substituted alkylene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein k is 1,2 or 3) ,-S (O)k(alkylene Base is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene Or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')- (alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene is substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(S) N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N =,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each independently H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
RaIt is to be each independently selected from by H, halogen, alkyl, substituted alkyl ,-N (R ')2、-C(O)k(wherein k is 1,2 to R ' Or 3) ,-C (O) N (R ')2,-OR ' and-S (O)kThe group of R ' compositions, wherein R ' are each independently H, alkyl or are substituted alkane Base.
In addition, including following amino acid：
Wherein described compound Optionally amino through protection, carboxyl through protection, or for its salt.In addition, any one of following alpha-non-natural amino acid all may be incorporated into In non-natural amino acid polypeptides.
In addition, including having the amino acid of the structure of formula (VII) below：
Wherein：
B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon alkylene Base, lower, low-carbon alkenylene are substituted, the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon is substituted, is substituted the sub- miscellaneous alkane of low-carbon Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein K be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
RaIt is each independently selected from by H, halogen, alkyl, substituted alkyl ,-N (R ')2、-C(O)kR ' (wherein k be 1,2 or 3)、-C(O)N(R′)2,-OR ' and-S (O)kThe group of R ' compositions, wherein R ' are each independently H, alkyl or substituted alkyl； And n is 0 to 8；
Condition is when A is-(CH2)4- when, B is not -NHC (O) (CH2CH2)-。
In addition, including following amino acid：
Wherein described compound optionally amino through protection, optionally carboxyl through protection, optionally amino through protection And carboxyl is also through protection, or it is its salt.In addition, any one of these alpha-non-natural amino acids and following alpha-non-natural amino acid All it may be incorporated into non-natural amino acid polypeptides.
In addition, including having the amino acid of the structure of formula (VIII) below：
Wherein A is optional, and when it is present for lower, be substituted lower, low-carbon cycloalkylidene, Be substituted low-carbon cycloalkylidene, low-carbon alkenylene, be substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, be substituted it is sub- miscellaneous The sub- Heterocyclylalkyl of alkyl, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Inferior heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon alkylene Base, lower, low-carbon alkenylene are substituted, the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon is substituted, is substituted the sub- miscellaneous alkane of low-carbon Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein K be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present.In addition, Including having the amino acid of the structure of formula (IX) below：
B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon alkylene Base, lower, low-carbon alkenylene are substituted, the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon is substituted, is substituted the sub- miscellaneous alkane of low-carbon Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein K be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
Wherein RaIt is each independently selected from by H, halogen, alkyl, substituted alkyl ,-N (R ')2、-C(O)k(wherein k is R ' 1st, 2 or 3) ,-C (O) N (R ')2,-OR ' and-S (O)kThe group of R ' compositions, wherein R ' are each independently H, alkyl or are substituted Alkyl.
In addition, including following amino acid：
It is wherein described Compound optionally amino through protection, optionally carboxyl is through protection, optionally amino, through protection and carboxyl also through protection, or is Its salt.In addition, any one of these alpha-non-natural amino acids and following alpha-non-natural amino acid all may be incorporated into alpha-non-natural amino acid In polypeptide.
In addition, including having the amino acid of the structure of formula (X) below：
Wherein B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon is sub- Alkyl, be substituted lower, low-carbon alkenylene, be substituted the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon, to be substituted low-carbon Asia miscellaneous Alkyl ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (its Middle k be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
RaIt is each independently selected from by H, halogen, alkyl, substituted alkyl ,-N (R ')2、-C(O)kR ' (wherein k be 1,2 or 3)、-C(O)N(R′)2,-OR ' and-S (O)kThe group of R ' compositions, wherein R ' are each independently H, alkyl or substituted alkyl； And n is 0 to 8.
In addition, including following amino acid：
Wherein described compound optionally amino through protection, optionally carboxyl through protection, optionally amino through protect Protect and carboxyl is also through protection, or be its salt.In addition, any in these alpha-non-natural amino acids and following alpha-non-natural amino acid Person may be incorporated into non-natural amino acid polypeptides.
In addition to mono carbonyl structure, alpha-non-natural amino acid specifically described herein may also comprise such as dicarbapentaborane, dicarbapentaborane sample Group, masked dicarbapentaborane and through protecting the group such as dicarbapentaborane.
For example, including below there is the amino acid of the structure of formula (XI)：
Wherein A is optional, and when it is present for lower, be substituted lower, low-carbon cycloalkylidene, Be substituted low-carbon cycloalkylidene, low-carbon alkenylene, be substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, be substituted it is sub- miscellaneous The sub- Heterocyclylalkyl of alkyl, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Inferior heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon alkylene Base, lower, low-carbon alkenylene are substituted, the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon is substituted, is substituted the sub- miscellaneous alkane of low-carbon Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein K be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present.
In addition, including having the amino acid of the structure of formula (XII) below：
B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon alkylene Base, lower, low-carbon alkenylene are substituted, the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon is substituted, is substituted the sub- miscellaneous alkane of low-carbon Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (wherein K be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
Wherein RaIt is each independently selected from by H, halogen, alkyl, substituted alkyl ,-N (R ')2、-C(O)k(wherein k is R ' 1st, 2 or 3) ,-C (O) N (R ')2,-OR ' and-S (O)kThe group of R ' compositions, wherein R ' are each independently H, alkyl or are substituted Alkyl.
In addition, including following amino acid：
Wherein described compound optionally amino through protection, regarding feelings Condition carboxyl through protection, optionally amino through protection and carboxyl also through protection, or for its salt.In addition, these alpha-non-natural amino acids And any one of following alpha-non-natural amino acid all may be incorporated into non-natural amino acid polypeptides.
In addition, including having the amino acid of the structure of formula (XIII) below：
Wherein B is optional, and is the connexon selected from the group being made up of following group when it is present：Low-carbon is sub- Alkyl, be substituted lower, low-carbon alkenylene, be substituted the sub- miscellaneous alkyl of low-carbon alkenylene, low-carbon, to be substituted low-carbon Asia miscellaneous Alkyl ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)k- (its Middle k be 1,2 or 3) ,-S (O)k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene is substituted alkylene Base)-,-C (O) N (R ')-,-CON (R ')-(alkylidene is substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene Or be substituted alkylidene)-,-N (R ') CO- (alkylidene is substituted alkylidene)-,-N (R ') C (O) O- ,-S (O)kN(R′)-、- N(R′)C(O)N(R′)-、-N(R′)C(S)N(R′)-、-N(R′)S(O)kN (R ')-,-N (R ')-N=,-C (R ')=N- ,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ')2- N=N- and-C (R ')2- N (R ')-N (R ')-, wherein R ' is each only It is on the spot H, alkyl or substituted alkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
RaIt is each independently selected from by H, halogen, alkyl, substituted alkyl ,-N (R ')2、-C(O)kR ' (wherein k be 1,2 or 3)、-C(O)N(R′)2,-OR ' and-S (O)kThe group of R ' compositions, wherein R ' are each independently H, alkyl or substituted alkyl； And n is 0 to 8.
In addition, including following amino acid：
Wherein described compound optionally amino through protection, optionally carboxyl through protection, optionally Amino is through protection and carboxyl also through protection, or for its salt.In addition, these alpha-non-natural amino acids and following alpha-non-natural amino acid Any one of all may be incorporated into non-natural amino acid polypeptides.
In addition, including having the amino acid of the structure of formula (XIV) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
X1For C, S or S (O)；And L is alkylidene, is substituted alkylidene, N (R ') (alkylidene) or N (R ') and (is substituted Alkylidene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl.
In addition, including having the amino acid of the structure of formula (XIV-A) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
L is alkylidene, be substituted alkylidene, N (R ') (alkylidene) or N (R ') (being substituted alkylidene), wherein R ' be H, Alkyl, substituted alkyl, cycloalkyl are substituted cycloalkyl.
In addition, including having the amino acid of the structure of formula (XIV-B) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
L is alkylidene, be substituted alkylidene, N (R ') (alkylidene) or N (R ') (being substituted alkylidene), wherein R ' be H, Alkyl, substituted alkyl, cycloalkyl are substituted cycloalkyl.
In addition, including having the amino acid of the structure of formula (XV) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
X1For C, S or S (O)；And n is 0,1,2,3,4 or 5；And each CR8R9R on group8And R9It is independently of one another Selected from the group being made up of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8And R9=O or ring can be collectively formed Alkyl, or any two adjacent R8Group can collectively form cycloalkyl.
In addition, including having the amino acid of the structure of formula (XV-A) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；
And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
N is 0,1,2,3,4 or 5；And each CR8R9R on group8And R9It is each independently selected from by H, alkoxy, alkyl Amine, halogen, alkyl, the group of aryl composition, or any R8And R9It can collectively form=O or cycloalkyl, or any two is adjacent R8Group can collectively form cycloalkyl.
In addition, including having the amino acid of the structure of formula (XV-B) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
N is 0,1,2,3,4 or 5；And each CR8R9R on group8And R9It is each independently selected from by H, alkoxy, alkyl Amine, halogen, alkyl, the group of aryl composition, or any R8And R9It can collectively form=O or cycloalkyl, or any two is adjacent R8Group can collectively form cycloalkyl.
In addition, including having the amino acid of the structure of formula (XVI) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
X1For C, S or S (O)；And L is alkylidene, is substituted alkylidene, N (R ') (alkylidene) or N (R ') and (is substituted Alkylidene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl.
In addition, including having the amino acid of the structure of formula (XVI-A) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
L is alkylidene, be substituted alkylidene, N (R ') (alkylidene) or N (R ') (being substituted alkylidene), wherein R ' be H, Alkyl, substituted alkyl, cycloalkyl are substituted cycloalkyl.
In addition, including having the amino acid of the structure of formula (XVI-B) below：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；
R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
L is alkylidene, be substituted alkylidene, N (R ') (alkylidene) or N (R ') (being substituted alkylidene), wherein R ' be H, Alkyl, substituted alkyl, cycloalkyl are substituted cycloalkyl.
In addition, including the amino acid of the structure with formula (XVII)：
Wherein：
A is optional, and is when it is present lower, is substituted lower, low-carbon cycloalkylidene, through taking For low-carbon cycloalkylidene, low-carbon alkenylene, it is substituted the sub- miscellaneous alkyl of low-carbon alkenylene, alkynylene, low-carbon, is substituted sub- miscellaneous alkane The sub- Heterocyclylalkyl of base, low-carbon, low-carbon sub- Heterocyclylalkyl, arlydene are substituted, arlydene, inferior heteroaryl is substituted, is substituted Asia Heteroaryl, alkarylene, it is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；M is-C (R3)-、 Wherein (a) is represented and A groups Bond, and (b) is represented and respective carbonyl is bonded, R3And R4Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl Or it is substituted cycloalkyl, or R3And R4Or two R3Group or two R4Group optionally forms cycloalkyl or Heterocyclylalkyl；
R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
T3For key, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkanes Base；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present.
In addition, including the amino acid of the structure with formula (XVIII)：
Wherein：
M is-C (R3)-、 Wherein (a) is represented and A groups Bond, and (b) is represented and respective carbonyl is bonded, R3And R4Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl Or it is substituted cycloalkyl, or R3And R4Or two R3Group or two R4Group optionally forms cycloalkyl or Heterocyclylalkyl；
R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；
T3For key, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkanes Base；
R1It is optional, and is H, amino protecting group, resin, amino acid, polypeptide or polynucleotides when it is present；And
R2It is optional, and is OH, ester protection group, resin, amino acid, polypeptide or polynucleotides when it is present；
RaIt is to be each independently selected from by H, halogen, alkyl, substituted alkyl ,-N (R ')2、-C(O)kR ' (wherein k be 1, 2 or 3) ,-C (O) N (R ')2,-OR ' and-S (O)kThe group of R ' compositions, wherein R ' are each independently H, alkyl or are substituted alkane Base.
In addition, including the amino acid of the structure with formula (XIX)：
Wherein：
R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；And
T3For O or S.
In addition, including the amino acid of the structure with formula (XX)：
Wherein：
R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl.
In addition, including having the amino acid of the structure of formula (XXI) below：
In certain embodiments, chemically modification comprising alpha-non-natural amino acid polypeptide with produce reactive carbonyl or Dicarbapentaborane functional group.For example, the aldehyde official that can be used for engagement reaction can be produced by the functional group with neighboring amine groups and hydroxyl Can group.When bioactive molecule is polypeptide, for example can be used N-terminal serine or threonine (its can normal presence, or can lead to Cross chemistry or enzymatic digestion and expose) use periodate to produce aldehyde functional group under the conditions of gentle oxicracking.For example, ginseng See Gaertner et al., Bioconjug.Chem.3：262-268(1992)；Geoghegan, K. and Stroh, J., Bioconiug.Chem.3：138-146(1992)；Gaertner et al., J.Biol.Chem.269：7224-7230(1994). However, known method is confined to the N-terminal amino acid of peptide or protein matter in art.
In the present invention, non-naturally encoded amino acids with adjacent hydroxyl groups and amino can as " masked " aldehyde function Group is incorporated in polypeptide.For example, 5- oxylysines carry the hydroxyl adjacent with ε amine.Reaction condition for producing aldehyde leads to Often it is related to the sodium metaperiodate for adding molar excess in a mild condition, to avoid that oxygen occurs at other sites in polypeptide Change.The pH value of oxidation reaction is typically about 7.0.Typical reaction, which is related to, adds about 1.5 molar excess into the cushioning liquid of polypeptide Sodium metaperiodate, then cultivated in dark surrounds about 10 minutes.Referring for example to U.S. Patent No. 6,423,685.
Carbonyl or dicarbapentaborane functional group can in a mild condition in aqueous with the reagent selective reaction containing azanol, from And it is bonded to form corresponding oxime stable in physiological conditions.For example, referring to Jencks, W.P., J.Am.Chem.Soc.81,475- 481(1959)；Shao, J. and Tam, J.P., J.Am.Chem.Soc.117：3893-3899(1995).In addition, carbonyl or two carbonyls The uniqueness reactivity of base allows to carry out selective modification in the case where there are other amino acid side chains.For example, referring to Cornish, V.W. et al., J.Am.Chem.Soc.118：8150-8151(1996)；Geoghegan, K.F. and Stroh, J.G., Bioconjug.Chem.3：138-146(1992)；Mahal, L.K. et al., Science 276：1125-1128 (1997)。
The structure of alpha-non-natural amino acid and synthesis：Amino acid containing azanol
U.S. provisional patent application cases the 60/638,418th are to be incorporated herein in entirety by reference.Therefore, it is beautiful V chapters (entitled " Non-natural Amino Acids " part Bs in state's temporary patent application case the 60/638,418th (entitled " Structure and Synthesis of Non-Natural Amino Acids：Hydroxylamine- Containing Amino Acids ") provided in disclosure be completely suitable for preparing, purify, characterizing and using herein Described in alpha-non-natural amino acid, the method for non-natural amino acid polypeptides and the non-natural amino acid polypeptides through modification, composition (including Formulas I-XXXV), technology and strategy, its level of application are presented in this article completely just as the disclosure.The U.S. is special Sharp publication No. 2006/0194256, No. 2006/0217532 and No. 2006/0217289 and entitled " Compositions containing, methods involving, and uses ofnon-natural amino acid S and polypeptides " WO 2006/069246 is also to be incorporated herein in entirety by reference.
The chemical synthesis of alpha-non-natural amino acid
A variety of alpha-non-natural amino acids suitable for the present invention are purchased from such as Sigma (USA) or Aldrich (Milwaukee, WI, USA).The alpha-non-natural amino acid that can not be bought is optionally as provided herein or such as various publication Provided in and synthesize, or synthesized using standard method known to those skilled in the art.On organic synthesis technology, example Such as referring to Fessendon and Fessendon,Organic Chemistry, (1982, second edition, Willard Grant Press, BostonMass.)；March,Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York)；And Carey and Sundberg,Advanced Organic Chemistry(the 3rd edition, A and part B, 1990, PlenumPress, New York).Describing other publication of alpha-non-natural amino acid synthesis includes (for example) entitled " In Vivoincorporation of Unnatural Amino Acids " WO 2002/085923；Matsoukas et al., (1995)J.Med.Chem.,38,4660-4669；King, F.E. and Kidd, D.A.A. (1949) A New Synthesis ofGlutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates,J.Chem.Soc,3315-3319；Friedman, O.M. and Chatterrji, R. (1959) Synthesis of Derivatives ofGlutamine as Model Substrates for Anti-Tumor Agents.J.Am.Chem.Soc.81,3750-3752；Craig, J.C. et al., (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-l-methylbutyl]amino] quinoline(Chloroquine).J.Org.Chem.53,1167-1170；Azoulay, M., Vilmont, M. and Frappier, F. (1991) Glutamine analogues asPotential Antimalarials, .Eur.J.Med.Chem.26,201-5；Koskinen, A.M.P. and Rapoport, H. (1989) Synthesis of 4- Substituted Prolines as Conformationally Constrained Amino AcidAnalogues.J.Org.Chem.54,1859-1866；Christie, B.D. and Rapoport, H. (1985) Synthesis ofOptically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of(+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization.J.Org.Chem.50：1239-1246；Barton et al., (1987) Synthesis of Novel alpha-Amino-Acids andDerivatives Using Radical Chemistry：Synthesis of L-and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid and Appropriate Unsaturated Derivatives.Tetrahedron43：4297-4308；And Subasinghe et al., (1992) Quisqualic acid analogues：synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site.J.Med.Chem.35：4602-7.Referring also to entitled " Protein Arrays " U.S. Patent Publication case US No. 2004/0198637, it is to be incorporated herein by reference.
A. carbonyl reaction group
Amino acid with carbonyl reaction group allows a variety of by nucleophilic addition or aldol reaction connection molecule The reaction of (including but not limited to PEG or other water soluble molecules).
The exemplary amino acid containing carbonyl can be expressed as below：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl or substituted aryl；R2For H, alkyl, aryl, through taking Substituted alkyl and substituted aryl；And R3For H, amino acid, polypeptide or amino terminal modification group, and R4For H, amino acid, many Peptide or carboxyl terminal modification group.In certain embodiments, n is 1, R1For phenyl and R2For simple alkyl (that is, methyl, ethyl Or propyl group), and ketone part is located relative to the contraposition of alkyl side chain.In certain embodiments, n is 1, R1For phenyl and R2 For simple alkyl (that is, methyl, ethyl or propyl group), and ketone part is located relative to the meta of alkyl side chain.
Synthesis to acetyl group-(+/-)-phenylalanine and an acetyl group-(+/-)-phenylalanine is described in Zhang, Z. Et al., Biochemistry 42：In 6735-6746 (2003), it is to be incorporated herein by reference.Art Other amino acid containing carbonyl can be similarly prepared in technical staff.
In certain embodiments, the polypeptide comprising non-naturally encoded amino acids is chemically modified to produce reactive carbonyl Base functional group.For example, the aldehyde functional group that can be used for engagement reaction can be produced by the functional group with neighboring amine groups and hydroxyl. When bioactive molecule is polypeptide, for example can be used N-terminal serine or threonine (its can normal presence, or can pass through and change Learn or enzymatic digestion and expose) using periodate produce aldehyde functional group under the conditions of gentle oxicracking.For example, referring to Gaertner et al., Bioconjug.Chem.3：262-268(1992)；Geoghegan, K. and Stroh, J., Bioconjug.Chem.3：138-146(1992)；Gaertner et al., J.Biol.Chem.269：7224-7230(1994). However, known method is confined to the N-terminal amino acid of peptide or protein matter in art.
In the present invention, non-naturally encoded amino acids with adjacent hydroxyl groups and amino can as " masked " aldehyde function Group is incorporated in polypeptide.For example, 5- oxylysines carry the hydroxyl adjacent with ε amine.Reaction condition for producing aldehyde leads to Often it is related to the sodium metaperiodate for adding molar excess in a mild condition, to avoid that oxygen occurs at other sites in polypeptide Change.The pH value of oxidation reaction is typically about 7.0.Typical reaction, which is related to, adds about 1.5 molar excess into the cushioning liquid of polypeptide Sodium metaperiodate, then cultivated in dark surrounds about 10 minutes.Referring for example to U.S. Patent No. 6,423,685, it is It is incorporated herein by reference.
Carbonyl functional group can select with the reagent containing hydrazine, hydrazides, azanol or semicarbazides in aqueous in a mild condition Property reaction, it is bonded to be respectively formed under physiological condition stable corresponding hydrazone, oxime or semicarbazones (semicarbazone).Example Such as, referring to Jencks, W.P., J.Am.Chem.Soc.81,475-481 (1959)；Shao, J. and Tam, J.P., J.Am.Chem.Soc.117：3893-3899(1995).In addition, the uniqueness reactivity of carbonyl allows the presence of other amino acid sides Selective modification is carried out in the case of chain.For example, referring to Cornish, V.W. et al., J.Am.Chem.Soc.118：8150- 8151(1996)；Geoghegan, K.F. and Stroh, J.G., Bioconjug.Chem.3：138-146(1992)；Mahal, L.K. et al., Science 276：1125-1128(1997).
B. hydrazine, hydrazides or semicarbazides reactive group
Non-naturally encoded amino acids containing nucleophilic group (such as hydrazine, hydrazides or semicarbazides) allow and a variety of electrophilic subbases Group's reaction forms binding element (including but not limited to being reacted with PEG or other water-soluble polymers).
The exemplary amino acid containing hydrazine, hydrazides or semicarbazides can be expressed as below：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl or substituted aryl or it is not present；X be O, N or S or It is not present；R2For H, amino acid, polypeptide or amino terminal modification group, and R3Modified for H, amino acid, polypeptide or carboxyl terminal Group.
In certain embodiments, n is 4, R1It is not present and X is N.In certain embodiments, n is 2, R1It is not present and X It is not present.In certain embodiments, n is 1, R1For phenyl, X is O, and oxygen atom is located at pair of aliphatic group on aromatic ring Position.
Amino acid containing hydrazides, hydrazine and semicarbazides is available from commercial source.For example, Pidolidone-γ-hydrazides can be obtained From Sigma Chemical (St.Louis, MO).The other amino acid that can not be bought can be prepared by those skilled in the art. Referring for example to U.S. Patent No. 6,281,211, it is to be incorporated herein by reference.
Containing the polypeptide with hydrazides, hydrazine or the non-naturally encoded amino acids of semicarbazide function can with it is a variety of containing aldehyde or Molecule with similar chemically reactive other functional groups effectively and selectively reacts.For example, referring to Shao, J. and Tam, J., J.Am.Chem.Soc.117：3893-3899(1995).With nucleophilic group present on 20 kinds of common amino acids (including (but It is not limited to) serine or the hydroxyl of threonine or the amino of lysine and N-terminal) compare, hydrazides, hydrazine and semicarbazide function Unique reactivity make it to the reactive significantly stronger of aldehyde, ketone and other electrophilic groups.
C. the amino acid containing aminooxy group
Non-naturally encoded amino acids containing aminooxy group (also referred to as azanol) allow to react shape with a variety of electrophilic groups (including but not limited to reacted into binding element with PEG or other water-soluble polymers).Such as hydrazine, hydrazides and semicarbazides one Sample, the enhanced nucleophilicity of aminooxy group makes itself and a variety of molecules containing aldehyde or with similar chemically reactive other functional groups Effectively and selectively react.For example, referring to Shao, J. and Tam, J., J.Am.Chem.Soc.117：3893-3899 (1995)；H.Hang and C.Bertozzi, Acc.Chem.Res.34：727-736(2001).Although the knot with hydrazine radical reaction Fruit is corresponding hydrazone, but aminooxy group and the reaction of carbonyl containing-group (such as ketone) generally produce oxime.
The exemplary amino acid containing aminooxy group can be expressed as below：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl or substituted aryl or it is not present；X is for O, N, S or not In the presence of；M is 0-10；Y=C (O) is not present；R2For H, amino acid, polypeptide or amino terminal modification group, and R3For H, ammonia Base acid, polypeptide or carboxyl terminal modification group.In certain embodiments, n is 1, R1For phenyl, X is O, and m is 1, and Y is present. In certain embodiments, n is 2, R1It is not present with X, m is 0, and Y is not present.
Amino acid containing aminooxy group can be by readily available amino acid precursor (homoserine, serine and threonine) Prepare.For example, referring to M.Carrasco and R.Brown, J.Org.Chem.68：8853-8858(2003).It is some to contain aminooxy group Amino acid (such as L-2- amino -4- (aminooxy group) butyric acid) from natural origin separation (Rosenthal, G., LifeSci.60：1635-1641(1997)).Other amino acid containing aminooxy group can be prepared by those skilled in the art.
D. azide and alkyne reaction group
Unique reactivity of azide and alkynes functional group makes its pole suitable for polypeptide and the selectivity of other biomolecule Modification.Organic azide (especially aliphatic azide) and alkynes are generally stable to common reactive electrochemical conditions. Specifically, side chain (that is, the R of azide and alkynes functional group all to visible 20 kinds of common amino acids in naturally occurring polypeptide Group) it is inert.However, when azide and ethynylene group close to when, its " spring pressurization (spring-loaded) " property shows Dew, and it produces corresponding triazole by Hughes's root [3+2] cycloaddition reaction selectivity and effectively reaction.For example, referring to Chin J. et al., Science 301：964-7(2003)；Wang, Q. et al., J.Am.Chem.Soc.125,3192-3193 (2003)；Chin, J.W. et al., J.Am.Chem.Soc.124：9026-9027(2002).
Because Hughes's root cycloaddition reaction is related to selective cycloaddition reaction (referring for example to Padwa, A., COMPREHENSIVE ORGANIC SYNTHESIS, volume 4, (Trost, B.M. are compiled, 1991), the 1069-1109 pages；Huisgen, R., 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (Padwa, A. are compiled, 1984), the 1-176 pages) rather than nucleophilic displacement of fluorine, so being incorporated to containing nitrine The non-naturally encoded amino acids of base and side chain containing alkynes make it that gained polypeptide is chosen at the position of non-naturally encoded amino acids Sex modification.Be related to FGF-21 polypeptides containing azido or containing alkynes cycloaddition reaction can at room temperature under aqueous conditions by Catalytic amount be used for by Cu (II) in-situ reducing for Cu (I) reducing agent in the presence of addition Cu (II) (be including but not limited in The CuSO of catalytic amount4Form) carry out.For example, referring to Wang, Q. et al., J.Am.Chem.Soc.125,3192-3193 (2003)；Tornoe, C.W. et al., J.Org.Chem.67：3057-3064(2002)；Rostovtsev et al., Angew.Chem.Int.Ed.41：2596-2599(2002).Exemplary reducing agent includes (including but is not limited to) ascorbic acid Salt, metallic copper, quinine (quinine), quinhydrones, vitamin K, glutathione, cysteine, Fe2+、Co2+And externally-applied potential.
In the case of some Hughes's root [3+2] cycloaddition reactions needed between azide and alkynes, FGF-21 polypeptides Include the water-soluble polymer that the non-naturally encoded amino acids and desire of alkynyl moiety are connected with amino acid and include azide moiety. Or, can also carry out backward reaction (that is, has azide moiety on amino acid and there is alkynes portion on water-soluble polymer Point).
Nitrine functional group can also select with the water-soluble polymer containing aryl ester and through the appropriate functionalization in aryl phosphine part React to selecting property bonded to produce acid amides.Aryl phosphine groups in-situ reducing azido, and gained amine then with immediate ester Key effecting reaction is to produce corresponding amides.Referring for example to E.Saxon and C.Bertozzi, Science 287,2007-2010 (2000).Amino acid containing azido can be alkyl azide (including but not limited to 2- amino -6- azido -1- caproic acids) Or aromatic yl azide (to azido-phenylalanine).
Exemplary water-soluble polymer containing aryl ester and phosphine part can be expressed as below：
Wherein X can be O, N, S or to be not present, and Ph is phenyl, W be water-soluble polymer and R can be H, alkyl, aryl, Substituted alkyl and substituted aryl.Exemplary R group includes but is not limited to-CH2、-C(CH3)3、-OR′、-NR′R″、- SR ' ,-halogen ,-C (O) R ' ,-CONR ' R " ,-S (O)2R′、-S(O)2NR ' R " ,-CN and-NO2.R ', R ", R " ' and R " " are each Hydrogen is independently referred to, be substituted or be unsubstituted miscellaneous alkyl, be substituted or is unsubstituted aryl (including but not limited to through 1-3 The aryl of individual halogen substitution), be substituted or be unsubstituted alkyl, alkoxy or thio alkoxy or aryl alkyl.For example, When the compound of the present invention includes more than one R group, each R group is independently chosen, and when exist R ', R ", R " ' and During one of R " " group above, these groups are equally chosen independently of one another.When R ' and R " are connected with same nitrogen-atoms When, it can combine to form 5 yuan, 6 yuan or 7 yuan of rings with nitrogen-atoms.For example ,-NR ' R " are intended to (but not limited to) 1- pyrroles Alkyl and 4- morpholinyls.According to the above-mentioned discussion to substituent, it will be understood by one of ordinary skill in the art that term " alkyl " is intended Group including including the carbon atom combined with the group in addition to hydrogen-based, such as alkylhalide group (including but not limited to-CF3With- CH2CF3) and acyl group (including but not limited to-C (O) CH3、-C(O)CF3、-C(O)CH2OCH3Deng).
Nitrine functional group can also select with the water-soluble polymer containing thioesters and through the appropriate functionalization in aryl phosphine part React bonded to produce acid amides to property.Aryl phosphine groups in-situ reducing azido, and gained amine is then effectively anti-with thioester bond Should be to produce corresponding amides.Exemplary water-soluble polymer containing thioesters and phosphine part can be expressed as below：
Wherein n is 1-10；X can be O, N, S or be not present that Ph is phenyl, and W is water-soluble polymer.
The exemplary amino acid containing alkynes can be expressed as below：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl or substituted aryl or it is not present；X is for O, N, S or not In the presence of；M is 0-10, R2For H, amino acid, polypeptide or amino terminal modification group, and R3For H, amino acid, polypeptide or carboxyl end Terminal modified group.In certain embodiments, n is 1, R1For phenyl, X is not present, and m is 0 and acetylene moiety is located relative to alkane The contraposition of base side chain.In certain embodiments, n is 1, R1For phenyl, X is O, and m is 1 and propargyloxy is located relative to alkane The contraposition (that is, O- propargyls-tyrosine) of base side chain.In certain embodiments, n is 1, R1It is not present with X and m is 0 (that is, alkynes Glycinate).
Amino acid containing alkynes is on sale on the market.For example, propargylglycine is purchased from Peptech (Burlington, MA).Or, the amino acid containing alkynes can be prepared according to standard method.For example, for example can be such as Deiters, A. et al., J.Am.Chem.Soc.125：Synthesized described in 11782-11783 (2003) to propargyloxy benzene Alanine, and can be such as Kayser, B. et al., Tetrahedron 53 (7)：4- is synthesized described in 2475-2484 (1997) Alkynyl-L-phenylalanine.Those skilled in the art can prepare other amino acid containing alkynes.
The exemplary amino acid containing azido can be expressed as below：
Wherein n is 0-10；R1For alkyl, aryl, substituted alkyl, substituted aryl or it is not present；X is for O, N, S or not In the presence of；M is 0-10；R2For H, amino acid, polypeptide or amino terminal modification group, and R3For H, amino acid, polypeptide or carboxyl end Terminal modified group.In certain embodiments, n is 1, R1For phenyl, X is not present, and m is 0 and azide moiety is located at alkyl side chain Contraposition.In certain embodiments, n is 0-4 and R1It is not present with X, and m=0.In certain embodiments, n is 1, R1For Phenyl, X is O, and m is the contraposition that 2 and β azido epoxy groups are located relative to alkyl side chain.
Amino acid containing azido is available from commercial source.For example, 4- azidophenylalanines are available from Chem- Impex International, Inc. (Wood Dale, IL)., can be relative for the amino acid containing azido that can not be bought Azido is easily prepared using standard method known to those skilled in the art, methods described includes but is not limited to By replacing suitable leaving group (including but not limited to halogen ion, methanesulfonate, tosylate) or by opening through suitable When the lactone of protection.Referring for example to March,Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York).
E. amineothiot reactive group
Unique reactivity of the amineothiot functional group of β substitutions makes it be extremely applicable to by forming thiazolidine come selectivity Modify the polypeptide containing aldehyde radical and other biomolecule.Referring for example to J.Shao and J.Tam, J.Am.Chem.Soc.1995,117 (14)3893-3899.In certain embodiments, the amineothiot amino acid of β substitutions may be incorporated into FGF-21 polypeptides and subsequent Reacted with the water-soluble polymer comprising aldehyde functional group.In certain embodiments, water-soluble polymer, medicine binding element or other Pay(useful) load can be coupled by forming thiazolidine with the FGF-21 polypeptides comprising the β amineothiot amino acid replaced.
F. other reactive groups
The other reactive groups and non-day that may be incorporated into described in following patent application case in the FGF-21 polypeptides of the present invention Right coded amino acid, the patent application case is all to be incorporated herein in entirety by reference：U.S. Patent Publication case No. 2006/0194256；U.S. Patent Publication case the 2006/0217532nd；U.S. Patent Publication case the 2006/0217289th； US provisional patent the 60/755,338th；US provisional patent the 60/755,711st；US provisional patent the 60/755th, No. 018；International application the PCT/US06/49397th；WO 2006/069246；US provisional patent the 60/743rd, No. 041；US provisional patent the 60/743,040th；International application the PCT/US06/47822nd；The U.S. is temporarily special Profit the 60/882,819th；US provisional patent the 60/882,500th；With US provisional patent the 60/870,594th.
The cellular uptake of alpha-non-natural amino acid
Cell is used to be incorporated in protein to the intake of alpha-non-natural amino acid to design and selecting and (include but is not limited to) The problem generally considered during alpha-non-natural amino acid.For example, the high charge density of a-amino acid shows these compounds Can not possibly be cell-permeable.Natural amino acid is absorbed to by eucaryon by the set of the movement system based on protein In cell.Can carry out quickly screening to analyze which alpha-non-natural amino acid (if present) is absorbed by cell.For example, referring to example Ru entitled, " (it is by reference to Protein Arrays " U.S. Patent Publication case US 2004/0198637 It is incorporated herein) and Liu, D.R. and Schultz, P.G. (1999) Progress toward the evolutionof an organism with an expanded genetic code.PNAS United States96：Poison in 4780-4785 Property calibrating.Although can easily analyze intake, non-natural amino of the design suitable for cellular uptake approach by various calibratings The alternative solution of acid in vivo produces the biosynthesis pathway of amino acid to provide.
The biosynthesis of alpha-non-natural amino acid
Many biosynthesis pathways are present in cell producing amino acid and other compounds.Nature (bag Include (but not limited to) cell) in the biological synthesis method of specific alpha-non-natural amino acid may be not present, but the present invention provides described Method.For example, non-day is optionally produced in host cell by adding new enzyme or changing existing host cell approach The biosynthesis pathway of right amino acid.Other new enzymes are optionally naturally occurring enzyme or the enzyme manually developed.For example, it is right The biosynthesis of amino phenylalanine is (such as in entitled " In vivo incorporation of unnatural Presented in example in aminoacids " WO 2002/085923) rely on the known enzyme from other organisms combination Addition.The gene can be introduced into eukaryotic by using the plasmid-transformed cells comprising the gene of these enzymes.When When being expressed in cell, the enzymatic route of compound needed for the gene provides synthesis.The example of the enzyme type optionally added It is provided in Examples below.Other enzyme sequences are found in (such as) Genbank.The enzyme manually developed is also optionally with phase Tongfang Formula is added in cell.The cell mechanism and resource of cell is manipulated in this way to produce alpha-non-natural amino acid.
A variety of methods, which can be used for producing, to be used in biosynthesis pathway or the novel enzymes for developing existing approach.Citing comes Say, optionally using including but not limited to as Maxygen, Inc. (can obtain) institute by WWW on maxygen.com The recurrence of exploitation recombinates to develop novel enzymes and approach.For example, referring to Stemmer (1994), Rapid evolutionof a Protein in vitro by DNA shuffling,Nature370(4)：389-391；And Stemmer, (1994), DNAshuffling by random fragmentation and reassembly：In vitro recombination For molecularevolution,Proc.Natl.Acad.Sci.USA., 91：10747-10751.Similarly, optionally will Genencor (can be existed by WWWgenencor.comIt is upper to obtain) DesignPath that is developedTMFor metabolic pathway engineering Transformation, the including but not limited to engineered approach that O- methyl-L-tyrosines are produced in cell.This technology uses new base Because (including but is not limited to) is via functional genomics and molecular evolution and designs differentiated gene) combination in host's life Existing approach is rebuild in object.Diversa companies (can be existed by WWWdiversa.comIt is upper to obtain) quick screening is also provided The technology of gene library and gene approach, so as to including but not limited to set up new way.
Generally, the alpha-non-natural amino acid produced by the biosynthesis pathway of the engineered mistake of the present invention is to be sufficient for Effective Protein synthesis (including but not limited to n cell amount), but not up to influence the concentration or consumption of other amino acid The concentration of the degree of cellular resources is produced to the greatest extent.The typical concentration in vivo produced in this way is about 10mM to about 0.05mM. After utilizing the plasmid-transformed cells comprising the gene for producing the enzyme needed for particular approach and producing alpha-non-natural amino acid, depending on Situation further optimizes the production of alpha-non-natural amino acid for ribosomal protein synthesis and cell growth using in vivo selecting It is raw.
Polypeptide with alpha-non-natural amino acid
Being incorporated to for alpha-non-natural amino acid can be carried out for many purposes, and the purpose includes but is not limited to adjust protein The change of structure and/or function；Change size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, protease target site it is accessible Property；Targeting moiety (including but not limited to, for protein array)；Add bioactive molecule；Connect polymer；Connection is put Penetrating property nucleic；Adjust serum half-life；Adjust tissue infiltration (such as tumour)；Adjust active transport；Adjust tissue, cell or device Official's specificity or distribution；Adjust immunogenicity；Regulatory protein enzyme resistance etc..Protein including alpha-non-natural amino acid, which can have, to be increased Strong or even brand-new catalysis or the reasonable matter of biological thing.For example, optionally by including non-natural amino in protein Acid improves following property：Toxicity, bio distribution, structural property, spectral quality, chemistry and/or spectrochemical property, catalysis energy Power, half-life period (including but not limited to serum half-life), the ability reacted with other molecules (including but not limited to covalently or It is non-covalent) etc..It is new that composition including the protein comprising at least one alpha-non-natural amino acid is applied to (including but is not limited to) Clever therapeutic agent, diagnosticum, catalyzing enzyme, industrial enzyme, associated proteins (including but is not limited to antibody) and (including but is not limited to) egg The research of white matter 26S Proteasome Structure and Function.Referring for example to Dougherty, (2000) Unnatural Amino Acids as Probes Of Protein Structure andFunction,Current Opinion in Chemical Biology, 4：645- 652。
In one aspect of the invention, composition includes at least one, and there is at least one (to include but is not limited at least two It is individual, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or More than ten) protein of alpha-non-natural amino acid.Alpha-non-natural amino acid may be the same or different, including but not limited in protein In may be present 1, more than 2,3,4,5,6,7,8,9 or 10 or 10 different locis, its comprising 1,2,3,4,5,6,7,8,9 or 10 or More than 10 different alpha-non-natural amino acids.On the other hand, composition includes at least one present in protein (but less than complete Portion) protein that replaces through alpha-non-natural amino acid of specific amino acids.For the given egg with more than one alpha-non-natural amino acid For white matter, alpha-non-natural amino acid may be the same or different (including but not limited to the protein may include two or two with Upper different types of alpha-non-natural amino acid, or may include two identical alpha-non-natural amino acids).For with the non-day of two or more For the given protein of right amino acid, alpha-non-natural amino acid can identical, different or for identical type multiple non-natural aminos The combination of the acid alpha-non-natural amino acid different from least one.
Of interest protein or polypeptide with least one alpha-non-natural amino acid for the present invention feature.The present invention The polypeptide or protein with least one alpha-non-natural amino acid produced including the use of the compositions and methods of the invention.Figuration Agent (including but not limited to pharmaceutically acceptable excipient) can also exist together with protein.
By the way that protein of interest or polypeptide, albumen are produced using at least one alpha-non-natural amino acid in eukaryotic Matter or polypeptide should generally include modifying after eukaryotic translation.In certain embodiments, protein includes at least one non-natural Amino acid and at least one posttranslational modification in vivo carried out by eukaryotic, wherein the posttranslational modification is not by original Nucleus is carried out.For example, posttranslational modification includes (including but is not limited to) acetylation, acylation, lipid-modified, palmityl Change, palmitate addition, phosphorylation, the bonded modification of glycolipid, glycosylation etc..On the one hand, posttranslational modification includes passing through GlcNAc- Asparagine is bonded to make oligosaccharides (including but not limited to (GlcNAc-Man)2- Man-GlcNAc-GlcNAc) connect with asparagine Connect.Referring to table 1, its list the N- connection oligosaccharides of eukaryotic protein some examples (also may be present it is other do not illustrate it is residual Base).On the other hand, posttranslational modification is included by GalNAc- serines or GalNAc- threonines are bonded or GlcNAc- ammonia Acid or GlcNAc- threonines it is bonded make oligosaccharides (including but not limited to Gal-GalNAc, Gal-GlcNAc etc.) and serine or Threonine is connected.
Table 1：Pass through the example of the oligosaccharides of the bonded connections of GlcNAc
On the other hand, posttranslational modification includes precursor and (includes but is not limited to calcitonin precursor, calcitonin gene phase Close peptide precursor, Pre Pro PTH (preproparathyroid hormone), preproinsulin, proinsulin, preceding Ah Melanocortins original (prepro-opiomelanocortin), POMC etc.) proteolysis processing, be assembled into many subunits Albumen or macromolecular assembling thing, translate in another site in cell and (including but not limited to translate such as endoplasmic reticulum, height In the organelles such as dictyosome (Golgi apparatus), core, lysosome, peroxisome, mitochondria, chloroplaset, vacuole, Or by secretory pathway).In certain embodiments, protein includes secretion or positioning sequence, epitope label, FLAG marks Label, polyhistidyl tags, GST fusions etc..
One advantage of alpha-non-natural amino acid is that it provides the other chemical parts that can be used for adding other molecules.This A little modifications in vivo can be carried out or in vitro carried out in eukaryotic or non-eukaryotic.Therefore, in certain embodiments, Posttranslational modification is carried out by alpha-non-natural amino acid.For example, posttranslational modification can be entered by nucleophilic-electrophilic reaction OK.Currently used for the selective modification of protein most of reactions be related to nucleophilic and electrophilic reaction collocation thing between it is covalent Key is formed, including but not limited to α-halogenatedketone and the reaction of histidine or cysteine side chain.In these cases, selectivity It is that the number and accessibility of nucleophilic residues are determined in protein., can be in vitro and living in the protein of the present invention In vivo using the bigger reaction of other selectivity, such as reaction of non-natural ketone group amino acid and hydrazides or aminooxy compound. For example, referring to Cornish et al., (1996)J.Am.Chem.Soc, 118：8150-8151；Mahal et al., (1997)Science, 276：1125-1128；Wang et al., (2001)Science292：498-500；Chin et al., (2002)J.Am.Chem.Soc.124：9026-9027；Chin et al., (2002)Proc.Natl.Acad.Sci..99：11020- 11024；Wang et al., (2003)Proc.Natl.Acad.Sci, 100：56-61；Zhang et al., (2003)Biochemistry, 42：6735-6746；And Chin et al., (2003)Science, 301：964-7, the document be all with The mode of reference is incorporated herein.This, which allows to use, includes the big of fluorogen, crosslinking agent, carbohydrate derivative and cytotoxic molecule Measure the substantially any protein of reagent selected marker.Referring also to entitled " Glycoprotein synthesis " United States Patent (USP) 6th, 927, No. 042, it is to be incorporated herein by reference.Including but not limited to carried out by azido amino acid Posttranslational modification also can be by Staudinger coupled reaction (Staudinger ligation) (including but not limited to three virtues Base phosphonate reagent) carry out.Referring for example to Kiick et al., (2002) Incorporation of azides intorecombinant Proteins for chemoselective modification by the Staudinger ligation,PNAS99： 19-24。
The present invention provides another extremely effective method of selective modification protein, and it is related to response selection codon And alpha-non-natural amino acid (including but not limited to containing azido or alkynyl moiety) heredity is incorporated in protein.Then, may be used By include but is not limited to Hughes's root [3+2] cycloaddition reaction (for example, see Padwa, A.,ComprehensiveOrganic Synthesis, volume 4, (1991) Trost, B.M. volumes, Pergamon, Oxford, the 1069-1109 pages；And Huisgen, R.,1,3-Dipolar Cycloaddition Chemistry, (1984) Padwa, A. volumes, Wiley, New York, 1- Page 176) modify these amino acid side chains with including but not limited to alkynyl or azido derivant respectively.Because the method is related to Cycloaddition rather than nucleophilic displacement of fluorine, it is possible to high selective modification protein.Can at room temperature under aqueous conditions by Cu (I) salt of catalytic amount is added in reactant mixture and this reaction is carried out with splendid regioselectivity (1,4 ＞ 1,5).For example join Tornoe et al. is seen, (2002)J.Org.Chem.67：3057-3064；And Rostovtsev et al., (2002)Angew.Chem.Int.Ed.41：2596-2599.Workable another method is with four cysteine bases on double arsenic compounds Member carries out ligand exchange, referring for example to Griffin et al., (1998)Science281：269-272.
The molecule that can be added to by [3+2] cycloaddition in the protein of the present invention includes spreading out with azido or alkynyl Biological substantially any molecule.These molecules include but is not limited to dyestuff, fluorogen, crosslinking agent, carbohydrate derivative, polymer (the including but not limited to derivative of polyethylene glycol), photocrosslinking agent, chemical toxicity compound, affinity marker, biotin spread out Biology, resin, bead, the second protein or polypeptide (or more), polynucleotides (including but not limited to DNA, RNA etc.), gold Belong to chelating agent, co-factor, aliphatic acid, carbohydrate etc..These molecules can add to the non-natural amino with alkynyl respectively Acid (including but not limited to propargyloxyphenylalanine) or alpha-non-natural amino acid with azido (including (but are not limited In) to azido-phenylalanine) in.
V. the in vivo generation of the FGF-21 polypeptides comprising non-genetic coding amino acid
It tRNA the and tRNA synzyme through modification can be used in vivo to produce the FGF-21 polypeptides of the present invention, added Into in the amino acid that can not be encoded into naturally occurring system or replace the amino acid.
For example, the method for producing tRNA and tRNA synzyme using the amino acid that can not be encoded in naturally occurring system It is to be described in U.S. Patent No. 7,045, No. 337 and the 7th, 083, No. 970, the patent is to be hereby incorporated herein by In.Therefore these methods are related to the endogenous synthetases and tRNA produced independently of translation system and worked (and is sometimes referred to as " orthogonal ") body translation.Generally, translation system includes orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synzyme (O-RS).Generally, O-RS in translation system it is preferential using at least one non-naturally-occurring amino acid make O-tRNA aminoacylations and O-tRNA recognizes at least one not by the selection codon of other tRNA identifications in system.Therefore, translation system responds warp knit The selection codon of code and by the protein produced by non-naturally encoded amino acids insertion system so that " substitution " amino acid Make it into a certain position of coded polypeptide.
A variety of orthogonal tRNA and aminoacyl for specific synthesizing amino acid to be inserted in polypeptide have been described in art TRNA synzyme, and it is commonly available in the present invention.For example, ketone group specificity O-tRNA/ aminoacyl-tRNAs are synthesized Enzyme is to be described in Wang, L. et al., Proc.Natl.Acad.Sci.USA 100：56-61 (2003) and Zhang, Z. et al., Biochem.42(22)：In 6735-6746 (2003).Exemplary O-RS or part thereof is by polynucleotide sequence coding and wrapped The amino acid sequence disclosed in U.S. Patent No. 7,045, No. 337 and the 7th, 083, No. 970 is included, the patent is each with reference Mode is incorporated herein.To the corresponding O-tRNA molecules that O-RS is used together also be described in U.S. Patent No. 7,045,337 and In 7th, 083, No. 970, the patent is to be incorporated herein by reference.O-tRNA/ aminoacyl-tRNA synthetases pair Other examples are described in WO 2005/007870, WO 2005/007624 and WO2005/019415.
The example of azido specificity O-tRNA/ aminoacyl-tRNA synthetase systems is described in Chin, J.W. et al., J, Am.Chem.Soc.124：In 9026-9027 (2002).Azido-L-Phe exemplary O-RS sequences are included (but do not limit In) nucleotide sequence SEQ as disclosed in U.S. Patent No. 7,083,970 (it is incorporated herein by reference) ID NO：14-16 and 29-32 and amino acid sequence SEQ ID NO：46-48 and 61-64.It is exemplary suitable for the present invention O-tRNA sequences are including but not limited to such as institute in U.S. Patent No. 7,083,970 (it is incorporated herein by reference) The nucleotide sequence SEQ ID NO of announcement：1-3.Have to specific non-naturally encoded amino acids specific O-tRNA/ aminoacyls- Other examples of tRNA synzyme pair are described in U.S. Patent No. 7,045,337 (it is incorporated herein by reference) In.The O-RS and O-tRNA that ketone group containing and the amino acid containing azido are incorporated in saccharomyces cerevisiae are described in Chin, J.W. et al., Science 301：In 964-967 (2003).
Report several other orthogonal right.Describe for alpha-non-natural amino acid to be potentially incorporated in Escherichia coli Glutaminyl from saccharomyces cerevisiae tRNA and synzyme is (referring for example to Liu, D.R. and Schultz, P.G. (1999)Proc.Natl.Acad.Sci.U.S.A.96：4780-4785), aspartyl (referring for example to Pastrnak, M. et al., (2000)Helv.Chim.Acta83：2277-2286) with tyrosyl- (referring for example to Ohno, S. et al., (1998)J.Biochem. (Tokyo, Jpn.)124：1065-1068；And Kowal, A.K. et al., (2001)Proc.Natl.Acad.Sci.U.S.A.98：2268-2273) system.Describe to derive from Escherichia coli for saccharomyces cerevisiae Glutaminyl is (referring for example to Kowal, A.K. et al., (2001)Proc.Natl.Acad.Sci.U.S.A.98：2268- 2273) with tyrosyl- (referring for example to Edwards, H. and Schimmel, P. (1990)Mol.Cell.Biol.10：1633- 1641) system of synzyme.Escherichia coli tyrosyl- system has been used in vivo being incorporated to the iodo- L- of 3- in mammalian cell Tyrosine.Referring to Sakamoto.K.. et al., (2002)Nucleic Acids Res.30：4692-4699.
The use of O-tRNA/ aminoacyl-tRNA synthetases is related to the specific cryptosystem of selection coding non-naturally encoded amino acids Son.Although any codon can be used, selection is it is generally desirable in the cell of expression O-tRNA/ aminoacyl-tRNA synthetases Seldom or from untapped codon.For example, exemplary codon includes nonsense codon (such as terminator codon (amber Amber, ochre and opal)), the codon of four or more base and be rarely employed or original other natural Three base codons.
Can be used known method of mutagenesis in art (including but not limited to site-specific mutagenesis, cassette mutagenesis, Limitation selection mutagenesis etc.) specific selection codon is introduced into the appropriate location of FGF-21 polynucleotide encoding sequences.
Component (such as O- for producing the Protein synthesis mechanism that can be used for being incorporated to non-naturally encoded amino acids RS, O-tRNA and orthogonal O-tRNA/O-RS to) method be described in Wang, L. et al., Science 292：498-500 (2001)；Chin, J.W. et al., J. Am.Chem.Soc.124：9026-9027(2002)；Zhang, Z. et al., Biochemistry 42：In 6735-6746 (2003).Method and combination for being in vivo incorporated to non-naturally encoded amino acids Thing is described in U.S. Patent No. 7,045,337, and the patent is to be incorporated herein by reference.For selecting to be used for The method of orthogonal tRNA-tRNA synzyme pair in the in vivo translation system of organism is also described in U.S. Patent No. 7,045, In No. 337 and U.S. Patent No. 7,083,970, the patent is to be incorporated herein by reference.Entitled " Site Specific Incorporation of Keto Amino Acids into Proteins " PCT Publication case WO04/ No. 035743 (it is to be incorporated herein in entirety by reference) describes the orthogonal RS and tRNA for being incorporated to ketone group amino acid It is right.Entitled " Expanding the Eukaryotic Genetic Code " No. WO04/094593 (its of PCT Publication case It is to be incorporated herein in entirety by reference) describe to be incorporated to the orthogonal RS of non-naturally encoded amino acids in eukaryotic host cell With tRNA pairs.
Method for producing at least one restructuring orthogonal aminoacyl-tRNA synzyme (O-RS) is included：(a) from the first life Object is produced from (being optionally mutated) RS of at least one aminoacyl-tRNA synthetase (RS) library, first life Object includes but is not limited to prokaryotes body, such as Methanococcus jannaschii, thermophilic hot autotrophic methane bacteria, Halophiles, large intestine bar The ancient bacterium of bacterium, hyperthermophilic, strong red-hot coccus, the ancient bacterium of extreme thermophilic, thermophilic spring life archeobacteria, Thermophilic Bacterium (T.thermophilus) etc.；Or most eukaryotes；(b) selection (and/or screening) in the library of RS (being optionally mutated RS) The member for making orthogonal tRNA (O-tRNA) aminoacylated in the case where there are non-naturally encoded amino acids and natural amino acid, from And the pond that activity (is optionally mutated) RS is provided；And/or (c) selects do not depositing (optionally by Solid phase) in the pond Preferentially make the aminoacylated active RS (being including but not limited to mutated RS) of O-tRNA in the case of non-naturally encoded amino acids, So as to provide at least one restructuring O-RS；Wherein described at least one restructuring O-RS preferentially makes O- using non-naturally encoded amino acids TRNA aminoacylations.
In one embodiment, RS is nonactive RS.Nonactive RS can be produced by being mutated active RS.Citing comes Say, can be by making at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6 or at least About 10 or more than 10 amino acid mutations are different aminoacids (including but not limited to alanine) to produce nonactive RS.
Known various technologies can be used in art to produce mutation RS library, the technology includes (but not limiting In) reasonable design based on protein tridimensional RS structures or in random or reasonable designing technique RS nucleotides mutagenesis. For example, can by mutation site-specific, random mutation, produce multifarious recombination mutation, chimeric construct, rationally set Known other methods produce mutation RS in meter and specifically described herein or art.
In one embodiment, selection (and/or screening) (including (but is not limited in the library of RS (being optionally mutated RS) In)) make the aminoacylated work of orthogonal tRNA (O-tRNA) in the case where there are non-naturally encoded amino acids and natural amino acid Property member includes：By positive selection or selection markers (including but not limited to antibiotics resistance gene etc.) and (being optionally mutated) RS library is introduced into multiple cells, its it is positives selection and/or selection markers comprising at least one selection codon (including (but not limited to) amber, ochre or opal codon)；The multiple cell is set to be grown in the case where there is selective agent；It is logical Cross and suppress at least one selection codon in positive selection or selection markers and there is selective agent and/or selective agent to differentiate In the case of survival (or display specific reaction) cell so that provide the pond containing active (being optionally mutated) RS through the positive Select the subset of cell.Selective agent and/or screening agent concentration can optionally be changed.
On the one hand, positive selectable marker be chloramphenicol (chloramphenicol) acetyltransferase (CAT) gene and In CAT genes, selection codon is Amber stop codon.Optionally, positive selectable marker be beta-lactam enzyme gene and In beta-lactam enzyme gene, selection codon is Amber stop codon.On the other hand, positive selection marker comprising fluorescence or Luminous selection markers or the selection markers (including but not limited to cell surface marker) based on compatibility.
In one embodiment, Solid phase or screening are excellent in the case of in the absence of non-naturally encoded amino acids in pond First include the aminoacylated active RS of O-tRNA (optionally mutant)：By Solid phase or selection markers with being selected from positive The pond that the activity selected or screened (is optionally mutated) RS is introduced into multiple cells of the second organism, wherein Solid phase or screening Mark (includes but is not limited to antibiotics resistance gene, it is mould that it includes but is not limited to chlorine comprising at least one selection codon Plain acetyltransferase (CAT) gene)；And differentiate and be supplemented with the of non-naturally encoded amino acids and selective agent or selective agent Survived in one culture medium or show specificity screening reaction but do not supplementing non-naturally encoded amino acids and selective agent or selective agent The second culture medium in can not survive or show the cell of specific reaction, so as to provide depositing with least one restructuring O-RS Living cells or screening cell.For example, CAT authentication schemes optionally serve as positive choosing in the determination of appropriate O-RS recombinants Select and/or negative screening.For example, optionally presence or absence of one or more non-naturally encoded amino acids In the case of replicate clone pool on the growth plate containing CAT (its comprising at least one selection codon).It is therefore contemplated that only containing There is the bacterium colony grown on the flat board of non-naturally encoded amino acids to contain restructuring O-RS.On the one hand, selection (and/or screening) agent is changed Concentration.In certain aspects, the first organism is different from the second organism.Therefore, the first organism and/or the second organism Optionally include：Prokaryotes, eucaryote, mammal, Escherichia coli, fungi, yeast, archeobacteria (archaebacterium), eubacteria (eubacterium), plant, insect, protist etc..In other embodiments, sieve Choosing mark includes fluorescence or luminous selection markers or the selection markers based on compatibility.
In another embodiment, screen or select (including but not limited to Solid phase) activity (optionally to dash forward in pond Becoming) RS includes：RS pond is mutated from positive selection step (b) isolating active；By Solid phase or selection markers and activity (depending on feelings Condition is mutated) RS pond is introduced into multiple cells of the second organism, and wherein Solid phase or selection markers include at least one choosing Select codon and (including but not limited to include toxicity markers' gene of at least one selection codon, it includes but is not limited to Ribalgilase barnase gene)；And differentiate the survival or aobvious in the first culture medium for do not supplement non-naturally encoded amino acids Show specificity screening reaction but can not be survived in the second culture medium for be supplemented with non-naturally encoded amino acids or show specificity Screen the cell of reaction, so as to provide survivaling cell with least one restructuring O-RS or screen cell, wherein it is described at least One kind restructuring O-RS has specificity to non-naturally encoded amino acids.On the one hand, at least one described selection codon includes about two Individual or two or more selection codon.These embodiments optionally may include at least one described selection codon comprising two or Two or more select codon situation, and the first organism it is different from the second organism (include but is not limited to each biology Stereoscopic situation be (including but is not limited to) prokaryotes, eucaryote, mammal, Escherichia coli, fungi, yeast, archeobacteria, Eubacteria, plant, insect, protist etc.) situation.In addition, some aspects, which include negative selection marker, includes ribonucleic acid The situation of enzyme barnase gene (it includes at least one selection codon).Other side is optionally included including selection markers The situation of fluorescence or luminous selection markers or the selection markers based on compatibility.In embodiment herein, screening and/or choosing Select the change for optionally including screening and/or select stringency.
In one embodiment, the method for producing at least one restructuring orthogonal aminoacyl-tRNA synzyme (O-RS) It can further include：(d) at least one restructuring O-RS is separated；(e) produce from least one restructuring O-RS's Second group of O-RS (optionally mutant)；And (f) repeat step (b) and (c), preferentially make O-tRNA aminoacyls until obtaining to include The mutation O-RS of the ability of base.Optionally, step (d)-(f) is repeated into (including but is not limited to) at least about twice.One side Face, can be produced from extremely by mutagenesis (including but not limited to random mutagenesis, site-specific mutagenesis, restructuring or its combination) A kind of few restructuring O-RS second group of mutation O-RS.
In the above-mentioned methods, selection/screening step (including but not limited to positive selection/screening step (b), negative choosing Select/screen step (c) or it is positive with Solid phase/screening step (b) and (c)) stringency optionally include changing selection/sieve Select stringency.In another embodiment, positive selection/screening step (b), Solid phase/screening step (c) or positive and negative Selection/screening step (b) is with (c) comprising reporter gene is used, and wherein reporter gene is by fluorescence-activated cell sorting (FACS) detect or wherein reporter gene is by luminous detection.Reporter gene is optionally presented in cell surface, bacteriophage Show first-class and selected according to the compatibility or catalytic activity for being related to non-naturally encoded amino acids or the like.In an implementation In example, mutation synzyme is to be presented in cell surface, bacteriophage to be presented first-class.
Method for producing the orthogonal tRNA (O-tRNA) of restructuring includes：(a) produced by the first organism from least A kind of mutation tRNA libraries of tRNA (including but not limited to suppressing silver tRNA)；(b) selected in the library (including (but not limited to) Solid phase) or screen in the case of in the absence of the RS from the first organism by from the second organism Aminoacylated (being optionally mutated) tRNA of aminoacyl-tRNA synthetase (RS), so as to provide tRNA's (optionally mutant) Pond；And (c) selects or screened orthogonal RS (O-RS) aminoacyl by introducing in the tRNA (optionally mutant) pond The member of change, so as to provide at least one restructuring O-tRNA；Wherein described at least one restructuring O-tRNA identification selection codons And can not effectively be recognized and preferentially aminoacylated by O-RS by the RS from the second organism.In certain embodiments, it is described At least one tRNA is inhibiting factor tRNA and/or comprising with natural and/or nonnatural base unique three base codon, Or be nonsense codon, rare codon, unnatural codons, the codon comprising at least four base, amber codon, reddish brown Stone codon or opal terminator codon.In one embodiment, restructuring O-tRNA has the improvement of orthogonality.It should be appreciated that In some embodiments, O-tRNA is without modification optionally from the second organism is introduced into the first organism.In various embodiments In, the first organism and the second organism are identical or different and optionally (are wrapped selected from (include but is not limited to) prokaryotes Include (but not limited to) Methanococcus jannaschii, thermophilic hot autotrophic methane bacteria, Escherichia coli, Halophiles etc.), eucaryote, lactation move Thing, fungi, yeast, archeobacteria, eubacteria, plant, insect, protist etc..In addition, restructuring tRNA is by non-naturally encoded ammonia Base acid is optionally aminoacylated, and wherein non-naturally encoded amino acids are that in vivo natural biological is synthesized or given birth to by genetically manipulated Thing is synthesized.Non-naturally encoded amino acids are optionally added to the growth medium of at least the first organism or the second organism In.
On the one hand, selection (including but not limited to Solid phase) or screening pass through aminoacyl-tRNA in the library Aminoacylated (being optionally mutated) tRNA (step (b)) of synzyme includes：By toxicity markers' gene and (being optionally mutated) TRNA libraries are introduced into multiple cells from the second organism, and wherein toxicity markers' gene includes at least one selection codon (or cause to produce gene necessary to toxic agents or the gene of inhibitor (static agent) or organism, wherein the mark Gene includes at least one selection codon)；And selection survivaling cell, wherein survivaling cell contains orthogonal comprising at least one TRNA or non-functional tRNA (being optionally mutated) tRNA pond.For example, can be by using compa-ratios cell density Examine and determine to select survivaling cell.
On the other hand, toxicity markers' gene may include two or more selection codons.In the another of methods described In individual embodiment, toxicity markers' gene is ribalgilase barnase gene, and wherein ribalgilase barnase gene is included At least one amber codon.Ribalgilase barnase gene optionally may include two or more amber codons.
In one embodiment, the orthogonal RS (O- that selection or screening pass through introducing in (being optionally mutated) tRNA pond RS) aminoacylated member may include：By positive selection or riddled basins and O-RS with (being optionally mutated) tRNA's Pond is introduced into multiple cells from the second organism, wherein positive marker genes comprising drug resistance gene, (it is included (but not It is limited to) beta-lactam enzyme gene, it includes at least one selection codon, for example, at least one Amber stop codon) or it is raw Gene or the gene that toxic agents are detoxified necessary to object；And differentiate and (including (but do not limit there is selective agent or selective agent In) antibiotic) in the case of the survival that grows or screening cell, so as to provide the cell pool with least one restructuring tRNA, Wherein described at least one restructuring tRNA be by O-RS it is aminoacylated and respond at least one described selection codon and by ammonia In the translation product that base acid insertion is encoded by positive marker genes.In another embodiment, selective agent and/or selective agent are changed Concentration.
Method for producing specific O-tRNA/O-RS pairs is provided.Methods described includes：(a) produced by the first organism At least one tRNA mutation tRNA library is come from from birth；(b) in the library Solid phase or screen in the absence of come It is aminoacylated by the aminoacyl-tRNA synthetase (RS) from the second organism in the case of the RS of the first organism (being optionally mutated) tRNA, so as to provide and (optionally be mutated) tRNA ponds；(c) selection or sieve in (being optionally mutated) tRNA ponds The aminoacylated members of the orthogonal RS (O-RS) introduced were gated, so as to provide at least one restructuring O-tRNA.Described at least one Plant restructuring O-tRNA identification selections codon and can not effectively be recognized and preferential by O-RS by the RS from the second organism It is aminoacylated.Methods described also includes (d) and produced by the 3rd organism from least one aminoacyl-tRNA synthetase (RS) (optionally be mutated) RS library；(e) select or screen there are non-naturally encoded amino acids in the mutation RS libraries The member aminoacylated O-tRNA with least one restructuring is preferentially made in the case of natural amino acid, so as to provide activity (being optionally mutated) RS ponds；(f) in the pond Solid phase or screen in the situation in the absence of non-naturally encoded amino acids At least one restructuring activity aminoacylated O-tRNA is preferentially set (to be optionally mutated) RS down, so that it is special to provide at least one Different in nature O-tRNA/O-RS pairs, wherein at least one described specific O-tRNA/O-RS to comprising at least one to non-naturally encoded Amino acid has specific restructuring O-RS and at least one restructuring O-tRNA.Including the specific O- produced by methods described TRNA/O-RS pairs.For example, specific O-tRNA/O-RS is to may include and (include but is not limited to) mutRNATyr- MutTyrRS is to (such as mutRNATyr-SS12TyrRS to), mutRNALeu-mutLeuRS to, mutRNAThr-mutThrRS To, mutRNAGlu-mutGluRS equity.In addition, methods described includes, first identical with the 3rd organism (it includes (but not limiting In) Methanococcus jannaschii) situation.
Also selection is included in the present invention for the orthogonal tRNA-tRNA synthesis in the in vivo translation system of the second organism The method of enzyme pair.Methods described includes：The aminoacyl-tRNA for separating or obtaining by marker gene, tRNA and from the first organism Synzyme (RS) is introduced into first group of cell from the second organism；Marker gene and tRNA are introduced and come from the second organism Replicating cell group in；And survivaling cell or screening display of the selection in replicating cell group in nonviable first group are special Opposite sex screening reaction but can not provide the cell of this reaction in replicating cell group, wherein first group with replicating cell group be Grown in the case of there is selective agent or selective agent, wherein survival or screening cell include the live body varus for the second organism Translate the orthogonal tRNA-tRNA synzyme pair in system.In one embodiment, compare and select or screen including in vivo complementary Calibrating.The concentration of selective agent or selective agent can be changed.
The organism of the present invention includes a variety of organisms and multiple combinations.For example, the first of the inventive method is biological Body may be identical or different with the second organism.In one embodiment, organism is optionally prokaryotes body, including (but Be not limited to) Methanococcus jannaschii, thermophilic hot autotrophic methane bacteria, Halophiles, Escherichia coli, the ancient bacterium of hyperthermophilic, strong red-hot coccus, Extreme thermophilic Gu bacterium, thermophilic spring life archeobacteria, Thermophilic Bacterium etc..Or, organism optionally includes most eukaryotes, including (but not limited to) plant (including but not limited to complicated plant, such as monocotyledon or dicotyledon), algae, primary life Thing, fungi (including but not limited to yeast etc.), animal (including but not limited to mammal, insect, arthropod etc.) etc.. In another embodiment, the second organism is prokaryotes body, including but not limited to Methanococcus jannaschii, thermophilic autotrophy first The ancient bacterium of alkane bacillus, Halophiles, Escherichia coli, hyperthermophilic, Halophiles, strong red-hot coccus, the ancient bacterium of extreme thermophilic, the life of thermophilic spring are ancient Bacterium, Thermophilic Bacterium etc..Or, the second organism can be most eukaryotes, including but not limited to yeast, zooblast, Plant cell, fungi, mammalian cell etc..In various embodiments, the first organism is different from the second organism.
VI. position of the non-naturally-occurring amino acid in FGF-21 polypeptides
The present invention, which covers, is incorporated to one or more non-naturally-occurring amino acid in FGF-21 polypeptides.Can by one or More than one non-naturally-occurring amino acid is incorporated to specific location without destroying polypeptide active.This can be by carrying out " conservative " substitution To realize, the substitution including but not limited to replaces hydrophobic amino acid with hydrophobic amino acid, uses huge 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor Huge amino acid, replace hydrophilic amino acid with hydrophilic amino acid and/or in active unwanted position insert non-natural There is amino acid.
A variety of biochemistries and structural approach can be used to select in FGF-21 polypeptides for non-naturally encoded amino acids substitution Required site.Those skilled in the art is it is clear that any position of polypeptide chain is suitable for selection to be incorporated to non-natural Coded amino acid, and select can be based on rationally designing or realize any purpose or without particular needs by randomly choosing to carry out The purpose wanted.The selection in required site can be used for producing the FGF-21 molecules with property needed for any or activity (including (but not Be limited to) activator, super-agonists, inverse agonist, antagonist, acceptor combination conditioning agent, receptor activity modulators), dimer Or polymer forms, do not change activity or property compared with natural molecule, or manipulate any physically or chemically (example of polypeptide Such as dissolubility, aggregation or stability).For example, can be used known point mutation analysis in art, Alanine-scanning or Homologue scan method is bioactivity desired position to differentiate in FGF-21 polypeptides.It is most important to FGF-21 bioactivity Residue, the residue relevant with medical stability, antibody antigen determine that base or acceptor or Heparin-binding residue can undergo mutation.It is beautiful State's patent No. 5,580,723, No. 5,834,250, No. 6,013,478, No. 6,428,954 and No. 6,451,561 (it is to be incorporated herein by reference) description differentiates that system is carried out in the active domain for influenceing polypeptide active by using target material The method for analyzing the 26S Proteasome Structure and Function of polypeptide (such as FGF-21).Except being identified as pair by alanine or homolog-scanning mutagenesis The visual polypeptide of residue outside the vital residue of bioactivity pursued it is required activity and be for non-naturally encoded amino The good candidate of acid substitution.Or, the institute through differentiating to be pursued the vital site of bioactivity also visual polypeptide Need activity and be the good candidate replaced for non-naturally encoded amino acids.Another alternative is by for each on polypeptide chain Simply continuously replaced using non-naturally encoded amino acids in position and observe the influence to polypeptide active.Art Technical staff it is clear that selection replaces for alpha-non-natural amino acid in any polypeptide any mode of position, technology or Method is suitable in the present invention.
As described in the example that is provided later in this specification, many numbers are collected using the method for presentation in present application According to, and have found and successfully test potential and beneficial mutational site.It was found that on mutant structure and activity its His information, in addition including in example not specific description on the information for some details allocated and/or tested, can also study and contain There is the information of FGF-21 polypeptides of missing to determine the protein domain that may allow to be replaced with non-naturally encoded amino acids. The FGF-21 regions for being responsible for combining FGF-21 acceptors can be differentiated using protease digestion and monoclonal antibody in a similar manner. Exclusion may not allow after the residue that non-naturally encoded amino acids replace, can research institute propose be substituted at each rest position Influence.Model can be produced according to the three-dimensional crystalline structure of other FGF family members and FGF receptor.Protein Data Bank (Protein Data Bank；PDB, is obtained by WWW on rcsb.org) it is containing protein and nucleic acid molecule The integrated data store of three-dimensional structure data.If three-dimensional structure data can not be obtained, then model can be set up to study polypeptide Two grades and tertiary structure.Therefore, those skilled in the art can easily differentiate what can be replaced through non-naturally encoded amino acids Amino acid position.
In certain embodiments, FGF-21 polypeptides of the invention include the protein domain for being located at the structure for not destroying polypeptide In one or more non-naturally-occurring amino acid.
It may be residue not to be covered in potential receptor binding domain to be incorporated to the exemplary residue of non-naturally encoded amino acids, can It can completely or partially expose in a solvent, with neighbouring residue there is minimum or hydrogen-free to be bonded interaction, may be minimally Influenceed, be likely located in one or more exposures by neighbouring reactive residue, may be one or more With the 2nd FGF-21 or other molecules or its fragment site arranged side by side, be likely to be at as according to being combined with acceptor or be not associated with or Be coupled with another bioactive molecule or the FGF-21 polypeptides that are not coupled it is three-dimensional, two grades, three or four structure predicted During height pliability or structure are in rigid region, or it may be adjusted by changing the pliability or rigidity of complete structure on demand Save FGF-21 in itself or the dimer comprising one or more FGF-21 or the conformation of polymer.
As differentiated by crystallography, FGF protein familieses have common β-three-lobed structure or β-laminated structure (Harmer et al., Biochemistry 43：629-640(2004)).Skilled artisan recognize that, it is this right FGF-21 analysis permits a determination which amino acid residue is residual compared to the amino acid being hidden in tertiary protein structure It is surface exposure for base.Therefore, it is this hair as the amino acid of surface exposed residue with non-naturally encoded amino acids substitution Bright one embodiment.
In certain embodiments, one or more non-naturally encoded amino acids are incorporated to any position in FGF-21 In：SEQ ID NO：1 1-181 or in SEQ ID NO：Corresponding amino acid in 2-7.In certain embodiments, one or one Individual above non-naturally encoded amino acids are incorporated in one or more positions in FGF-21 following position：Position 1 is (i.e., In N-terminal) before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、 50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、 75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、 100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、 119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、 138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、 157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、 176th, 177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO： Corresponding amino acid in 2-7).In certain embodiments, one or more non-naturally encoded amino acids are incorporated to FGF-21's In one or more positions in following position：10、52、117、126、131、162、87、77、83、72、69、79、91、 96th, 108 and 110 (SEQ IDNO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one or More than one non-naturally encoded amino acids is incorporated in one or more positions in FGF-21 following position：10、52、 77th, 117,126,131 and 162 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In some embodiments In, one or more non-naturally encoded amino acids are incorporated to one or more positions in FGF-21 following position In：87、77、83、72(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one Individual or more than one non-naturally encoded amino acids are incorporated in one or more positions in FGF-21 following position：69、 79th, 91,96,108 and 110 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In one embodiment, non-naturally-occurring amino acid is located at 91 positions (the SEQ ID NO in FGF-21：1 or SEQ ID NO：Corresponding amino acid in 2-7).In one embodiment, non-naturally-occurring amino acid is located at 131 in FGF-21 Position (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In one embodiment, non-naturally-occurring ammonia Base acid is located at 108 positions (the SEQ ID NO in FGF-21：1 or in SEQ ID NO：Corresponding amino acid in 2-7).At one In embodiment, non-naturally-occurring amino acid is located at 77 positions (the SEQ ID NO in FGF-21：1 or in SEQ ID NO：In 2-7 Corresponding amino acid).In one embodiment, non-naturally-occurring amino acid is located at 72 positions (the SEQ ID NO in FGF-21：1 Or in SEQ ID NO：Corresponding amino acid in 2-7).In one embodiment, non-naturally-occurring amino acid is located in FGF-21 87 positions (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In one embodiment, non-natural is deposited It is located at 86 positions (the SEQID NO in FGF-21 in amino acid：1 or in SEQ ID NO：Corresponding amino acid in 2-7).One In individual embodiment, non-naturally-occurring amino acid is located at 126 positions (the SEQ ID NO in FGF-21：1 or in SEQ ID NO：2-7 In corresponding amino acid).In one embodiment, non-naturally-occurring amino acid is located at 110 positions (the SEQ ID in FGF-21 NO：1 or in SEQID NO：Corresponding amino acid in 2-7).In one embodiment, non-naturally-occurring amino acid is located at FGF-21 In 83 positions (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In one embodiment, non-natural There is 146 positions (SEQ ID NO of the amino acid in FGF-21：1 or in SEQ ID NO：Corresponding amino acid in 2-7). In one embodiment, non-naturally-occurring amino acid is located at 135 positions (the SEQ ID NO in FGF-21：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In one embodiment, non-naturally-occurring amino acid is located at 96 position (SEQ in FGF-21 ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In one embodiment, non-naturally-occurring amino acid is located at 36 positions (SEQ ID NO in FGF-21：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, there is non-naturally-occurring amino acid at position 91 and deposited below one at position In another non-naturally-occurring amino acid：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13, 14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、 39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、 64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、 89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、 111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、 130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、 149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、 168th, 169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl of protein End) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, at position 91 There is non-naturally-occurring amino acid and there is another non-naturally-occurring amino acid at position below one：131、108、 77th, 72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding ammonia in 2-7 Base acid).In another embodiment, in position 91 and (the SEQ ID NO of position 131：1 or in SEQ ID NO：It is corresponding in 2-7 Amino acid) there is non-naturally-occurring amino acid in place.In another embodiment, in position 91 and (the SEQ ID NO of position 77：1 or In SEQ ID NO：Corresponding amino acid in 2-7) there is non-naturally-occurring amino acid in place.In another embodiment, in position 91 and (the SEQ ID NO of position 108：1 or in SEQ ID NO：Corresponding amino acid in 2-7) there is non-naturally-occurring amino in place Acid.In another embodiment, in position 131 and (the SEQ ID NO of position 108：1 or in SEQ IDNO：Corresponding ammonia in 2-7 Base acid) there is non-naturally-occurring amino acid in place.In another embodiment, in position 131 and (the SEQ ID NO of position 77：1 or In SEQ ID NO：Corresponding amino acid in 2-7) there is non-naturally-occurring amino acid in place.In another embodiment, in position 131(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7) there is non-naturally-occurring amino acid in place.Another In individual embodiment, in (the SEQ ID NO of position 108：1 or in SEQ ID NO：Corresponding amino acid in 2-7) there is non-natural in place There is amino acid.In another embodiment, in (the SEQ ID NO of position 77：1 or in SEQ ID NO：Corresponding amino in 2-7 Acid) there is non-naturally-occurring amino acid in place.In another embodiment, in (the SEQ ID NO of position 72：1 or in SEQ ID NO： Corresponding amino acid in 2-7) there is non-naturally-occurring amino acid in place.In another embodiment, in (the SEQ ID NO of position 87： 1 or in SEQ ID NO：Corresponding amino acid in 2-7) there is non-naturally-occurring amino acid in place.In another embodiment, in place Put 86 (SEQ IDNO：1 or in SEQ ID NO：Corresponding amino acid in 2-7) the connected non-naturally-occurring amino of place's presence Acid.In another embodiment, in (the SEQ ID NO of position 126：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place deposits In non-naturally-occurring amino acid.In another embodiment, in (the SEQ ID NO of position 110：1 or in SEQ ID NO：In 2-7 Corresponding amino acid) there is non-naturally-occurring amino acid in place.In another embodiment, in (the SEQ IDNO of position 83：1 or SEQ ID NO：Corresponding amino acid in 2-7) there is non-naturally-occurring amino acid in place.
In another embodiment, there is non-naturally-occurring amino acid and one in following position at position 91 Or there are one or more other non-naturally-occurring amino acid at more than one position：Before position 1 (that is, in N-terminal), 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、 29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、 54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、 79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、 103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、 122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、 141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、 160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、 179th, 180,181,182 (that is, in the carboxyl terminal of protein) (SEQ IDNO：1 or in SEQ ID NO：Corresponding ammonia in 2-7 Base acid).In another embodiment, exist at position 91 non-naturally-occurring amino acid and one in following position or There are one or more other non-naturally-occurring amino acid at more than one position：131、108、77、72、87、86、126、 110th, 83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, exist at position 131 non-naturally-occurring amino acid and in following position one There is another non-naturally-occurring amino acid at individual or more than one position：Before position 1 (that is, in N-terminal), 1,2,3,4,5, 6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、 33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、 58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、 83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、 106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、 125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、 144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、 163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、 182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ IDNO：Corresponding amino acid in 2-7).Another In individual embodiment, there are non-naturally-occurring amino acid and one or more positions in following position at position 131 Put place and there is another non-naturally-occurring amino acid：131st, 108,77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, exist at position 108 non-naturally-occurring amino acid and in following position two There are two or more other non-naturally-occurring amino acid at individual or two or more position：Position 1 (that is, in N-terminal) it Before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27, 28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、 53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、 78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、 102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、 121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、 140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、 159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、 178th, 179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ IDNO：1 or in SEQ ID NO：Phase in 2-7 Answer amino acid).In another embodiment, there is non-naturally-occurring amino acid at position 108 and in following position There are two or more other non-naturally-occurring amino acid at two or more positions：131、108、77、72、87、 86th, 126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, there is non-naturally-occurring amino acid and one in following position at position 77 Or there is another non-naturally-occurring amino acid at more than one position：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6, 7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、 33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、 58、59、60、61、62、63、64、65、66、61、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、 83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、 106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、 125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、 144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、 163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、 182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ IDNO：Corresponding amino acid in 2-7).Another In individual embodiment, there are non-naturally-occurring amino acid and one or more positions in following position at position 77 There is another non-naturally-occurring amino acid in place：131st, 108,77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
Research on the crystal structure of FGF-21 or FGF family members and itself and the interaction of FGF receptor can refer to Show which particular amino acid residue have can be completely or partially close to the side chain of solvent.Non-naturally encoded amino at these positions Sour side chain can be away from protein surface and entrance solvent.In certain embodiments, in these positions one or more Non-naturally-occurring amino acid at position is connected with water-soluble polymer, including but not limited to following position：Position 1 (that is, exists N-terminal) before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25, 26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、 51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、 76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、 101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、 120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、 139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、 158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、 177th, 178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：In 2-7 Corresponding amino acid).In certain embodiments, the non-naturally-occurring amino acid at a position connects with water-soluble polymer Connect, including following position：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16, 17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、 42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、 67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、 92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、 113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、 132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、 151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、 170th, 171,172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, two in these positions Or the non-naturally-occurring amino acid at two or more position is connected with water-soluble polymer, include but is not limited to following position： Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21, 22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、 47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、 72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、 97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、 117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、 136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、 155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、 174th, 175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, it is non-at one or more positions in these positions Naturally occurring amino acid is connected with water-soluble polymer, including but not limited to following position：10、52、117、126、131、 162nd, 87,77,83,72,69,79,91,96,108 and 110 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino in 2-7 Acid).In certain embodiments, the non-naturally-occurring amino acid at one or more positions in these positions and water solubility Polymer is connected：10、52、77、117、126、131、162(SEQ ID NO：1 or in SEQ IDNO：Corresponding amino in 2-7 Acid).In certain embodiments, the non-naturally-occurring amino acid at one or more positions in these positions and water solubility Polymer is connected：87、77、83、72(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In some implementations In example, the non-naturally-occurring amino acid at one or more positions in these positions is connected with water-soluble polymer：69、 79th, 91,96,108 and 110 (SEQ ID NO：1 or in SEQID NO：Corresponding amino acid in 2-7).In certain embodiments, The non-naturally-occurring amino acid at one or more positions in these positions is connected with water-soluble polymer：91、131、 108th, 77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Phase in 2-7 Answer amino acid).In another embodiment, when non-naturally-occurring amino acid is present in (the SEQ ID NO of amino acid 91：1 or SEQ ID NO：Corresponding amino acid in 2-7) place when, the non-naturally-occurring amino acid is connected with water-soluble polymer.
In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 91, And exist below one at position another non-naturally-occurring amino acid and these non-naturally-occurring amino acid with it is water-soluble Property polymer connection：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17, 18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、 43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、 68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、92、93、 94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、 114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、 133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、 152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、 171st, 172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, there is non-natural at position 91 to deposit There are one or more other non-naturally-occurring amino acid at position in amino acid and below one, and these are non- Naturally occurring amino acid is connected with water-soluble polymer：131st, 108,77,72,87,86,126,110,83,146,135,96 and 36(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, deposited at position 91 There are one or more at position in the non-naturally-occurring amino acid being connected with water-soluble polymer, and below one Other non-naturally-occurring amino acid and these non-naturally-occurring amino acid are connected with water-soluble polymer：Position 1 is (that is, at N ends End) before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、 52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、 77、78、79、80、81、82、83、84、85、86、87、88、89、90、92、93、94、95、96、97、98、99、100、101、 102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、 121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、 140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、 159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、 178th, 179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Phase in 2-7 Answer amino acid).In another embodiment, there is the non-naturally-occurring amino being connected with water-soluble polymer at position 91 Acid, and in the presence of other non-naturally-occurring amino acid that one or more are connected with water-soluble polymer：131、108、77、 72nd, 87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino in 2-7 Acid).In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 91, and At two or more positions in following position exist two or more other non-naturally-occurring amino acid and These non-naturally-occurring amino acid are connected with water-soluble polymer：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8, 9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、 35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、 60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、 85、86、87、88、89、90、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、 108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、 127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、 146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、 165th, 166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, exist The carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment In, there are the non-naturally-occurring amino acid being connected with water-soluble polymer, and two in following position at position 91 Or there are two or more other non-naturally-occurring amino acid and these non-naturally-occurring amino at two or more position Acid is connected with water-soluble polymer：131st, 108,77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO： 1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 131, And exist below one at position another non-naturally-occurring amino acid and these non-naturally-occurring amino acid with it is water-soluble Property polymer connection：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17, 18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、 43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、 68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、 93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、 114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、132、133、 134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、 153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、 172nd, 173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO： 1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, there is non-naturally-occurring at position 131 Amino acid, and below one at position exist one or more other non-naturally-occurring amino acid and these non-days So there is amino acid to be connected with water-soluble polymer：91st, 108,77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, exist at position 131 The non-naturally-occurring amino acid being connected with water-soluble polymer, and have one or more its at position below one Its non-naturally-occurring amino acid and these non-naturally-occurring amino acid are connected with water-soluble polymer：Position 1 is (that is, at N ends End) before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、 52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、 77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、 102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、 121、122、123、124、125、126、127、128、129、130、132、133、134、135、136、137、138、139、140、 141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、 160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、 179th, 180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding ammonia in 2-7 Base acid).In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 131, and And in the presence of other non-naturally-occurring amino acid that one or more are connected with water-soluble polymer：91、108、77、72、87、 86th, 126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).Another In one embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 131, and with bottom There are two or more other non-naturally-occurring amino acid and these non-days at two or more positions in putting So there is amino acid to be connected with water-soluble polymer：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11, 12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、 37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、 62、63、64、65、66、61、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、 87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、 109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、 128、129、130、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、 148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、 167th, 168,169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, in protein Carboxyl terminal) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, in position There is the non-naturally-occurring amino acid that is connected with water-soluble polymer at 131, and two or two in following position with Exist at upper position two or more other non-naturally-occurring amino acid and these non-naturally-occurring amino acid with it is water-soluble Property polymer connection：91st, 108,77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 108, And exist below one at position another non-naturally-occurring amino acid and these non-naturally-occurring amino acid with it is water-soluble Property polymer connection：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17, 18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、 43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、 68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、 93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、109、110、111、112、113、114、 115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、 134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、 153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、 172nd, 173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO： 1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, there is non-naturally-occurring at position 108 Amino acid, and below one at position exist one or more other non-naturally-occurring amino acid and these non-days So there is amino acid to be connected with water-soluble polymer：91st, 131,77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, exist at position 108 The non-naturally-occurring amino acid being connected with water-soluble polymer, and have one or more its at position below one Its non-naturally-occurring amino acid and these non-naturally-occurring amino acid are connected with water-soluble polymer：Position 1 is (that is, at N ends End) before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、 52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、61、68、69、70、71、72、73、74、75、76、 77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、 102、103、104、105、106、107、109、110、111、112、113、114、115、116、117、118、119、120、121、 122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、 141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、 160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、 179th, 180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding ammonia in 2-7 Base acid).In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 108, and And in the presence of other non-naturally-occurring amino acid that one or more are connected with water-soluble polymer：91、131、77、72、87、 86th, 126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).Another In one embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 108, and with bottom There are two or more other non-naturally-occurring amino acid and these non-days at two or more positions in putting So there is amino acid to be connected with water-soluble polymer：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11, 12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、 37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、 62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、 87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、109、 110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、 129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、 148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、 167th, 168,169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, in protein Carboxyl terminal) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, in position There is the non-naturally-occurring amino acid that is connected with water-soluble polymer at 108, and two or two in following position with Exist at upper position two or more other non-naturally-occurring amino acid and these non-naturally-occurring amino acid with it is water-soluble Property polymer connection：91st, 131,77,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 77, And exist below one at position another non-naturally-occurring amino acid and these non-naturally-occurring amino acid with it is water-soluble Property polymer connection：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17, 18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、 43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、 68、69、70、71、72、73、74、75、76、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、 94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、 114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、 133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、 152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、 171st, 172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, there is non-natural at position 77 to deposit There are one or more other non-naturally-occurring amino acid at position in amino acid and below one, and these are non- Naturally occurring amino acid is connected with water-soluble polymer：91st, 131,108,72,87,86,126,110,83,146,135,96 and 36(SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment, deposited at position 77 There are one or more at position in the non-naturally-occurring amino acid being connected with water-soluble polymer, and below one Other non-naturally-occurring amino acid and these non-naturally-occurring amino acid are connected with water-soluble polymer：Position 1 is (that is, at N ends End) before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、 52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、 78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、 102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、 121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、 140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、 159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、 178th, 179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Phase in 2-7 Answer amino acid).In another embodiment, there is the non-naturally-occurring amino being connected with water-soluble polymer at position 77 Acid, and in the presence of other non-naturally-occurring amino acid that one or more are connected with water-soluble polymer：91、131、108、 72nd, 87,86,126,110,83,146,135,96 and 36 (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino in 2-7 Acid).In another embodiment, there is the non-naturally-occurring amino acid being connected with water-soluble polymer at position 77, and At two or more positions in following position exist two or more other non-naturally-occurring amino acid and These non-naturally-occurring amino acid are connected with water-soluble polymer：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8, 9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、 35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、 60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、78、79、80、81、82、83、84、85、 86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、 108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、 127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、 146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、 165th, 166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, exist The carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In another embodiment In, there are the non-naturally-occurring amino acid being connected with water-soluble polymer, and two in following position at position 77 Or there are two or more other non-naturally-occurring amino acid and these non-naturally-occurring amino at two or more position Acid is connected with water-soluble polymer：91st, 131,108,72,87,86,126,110,83,146,135,96 and 36 (SEQ ID NO： 1 or in SEQ ID NO：Corresponding amino acid in 2-7).
In another embodiment, in position 91 and (the SEQ ID NO of position 131：1 or in SEQ ID NO：Phase in 2-7 Answer amino acid) there is one or more positions and water-soluble polymeric in non-naturally-occurring amino acid and these positions in place Thing is connected.In another embodiment, in position 91 and (the SEQ ID NO of position 77：1 or in SEQ ID NO：It is corresponding in 2-7 Amino acid) there is one or more positions and water-soluble polymer in non-naturally-occurring amino acid and these positions in place Connection.In another embodiment, in position 91 and (the SEQ ID NO of position 108：1 or in SEQ ID NO：It is corresponding in 2-7 Amino acid) there is one or more positions and water-soluble polymer in non-naturally-occurring amino acid and these positions in place Connection.In another embodiment, in position 131 and (the SEQ IDNO of position 108：1 or in SEQ ID NO：It is corresponding in 2-7 Amino acid) there is one or more positions and water-soluble polymer in non-naturally-occurring amino acid and these positions in place Connection.In another embodiment, in position 131 and (the SEQ ID NO of position 77：1 or in SEQ ID NO：It is corresponding in 2-7 Amino acid) there is one or more positions and water-soluble polymer in non-naturally-occurring amino acid and these positions in place Connection.In another embodiment, in (the SEQ ID NO of position 91：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place In the presence of the non-naturally-occurring amino acid being connected with water-soluble polymer.In another embodiment, in (the SEQ ID of position 131 NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7) there is the non-naturally-occurring amino that is connected with water-soluble polymer in place Acid.In another embodiment, in (the SEQ ID NO of position 108：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place deposits In the non-naturally-occurring amino acid being connected with water-soluble polymer.In another embodiment, in (the SEQ ID NO of position 77：1 Or in SEQ IDNO：Corresponding amino acid in 2-7) there is the non-naturally-occurring amino acid that is connected with water-soluble polymer in place. In another embodiment, in (the SEQ ID NO of position 72：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place exist and water The non-naturally-occurring amino acid of soluble polymer connection.In another embodiment, in (the SEQ ID NO of position 87：1 or in SEQ ID NO：Corresponding amino acid in 2-7) there is the non-naturally-occurring amino acid that is connected with water-soluble polymer in place.In another reality Apply in example, in (the SEQ ID NO of position 86：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place exist and water-soluble polymeric The non-naturally-occurring amino acid of thing connection.In another embodiment, in (the SEQ ID NO of position 126：1 or in SEQ ID NO： Corresponding amino acid in 2-7) there is the non-naturally-occurring amino acid that is connected with water-soluble polymer in place.In another embodiment In, in (the SEQ ID NO of position 110：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place exist and water-soluble polymer The non-naturally-occurring amino acid of connection.In another embodiment, in (the SEQ ID NO of position 83：1 or in SEQ ID NO：2-7 In corresponding amino acid) there is the non-naturally-occurring amino acid that is connected with water-soluble polymer in place.
The given position in FGF-21 polypeptides can be replaced with a variety of non-naturally encoded amino acids, or a variety of non-naturals can be compiled Code amino acid is incorporated in the given position in FGF-21 polypeptides.In general, according to FGF-21 polypeptides or other FGF families into Member selects the specific non-naturally encoded amino acids for being incorporated to the research of the three-dimensional crystalline structure of its acceptor, wherein it is preferred that conservative Substitution (that is, is taken with the non-naturally encoded amino acids (such as to acetyl phenyl alanine or O- propargyls tyrosine) based on aryl For Phe, Tyr or Trp) and the specificity engagement chemistry that wishes to introduce into FGF-21 polypeptides (for example, if necessary to using having The water-soluble polymer of alkynyl moiety realizes Xiu Gensi [3+2] cycloaddition, or needs using having aryl ester that (it is incorporated to phosphine portion again Point) water-soluble polymer form amido link, then introduce 4- azidophenylalanines).
In one embodiment, methods described further comprises alpha-non-natural amino acid being incorporated in protein, wherein described Alpha-non-natural amino acid includes the first reactive group；And make protein with comprising the second reactive group molecule (including (but It is not limited to) it is mark, dyestuff, polymer, water-soluble polymer, the derivative of polyethylene glycol, photocrosslinking agent, radionuclide, thin Cellular toxicity compound, medicine, affinity marker, photoaffinity mark, reactive compounds, resin, the second protein or polypeptide Or polypeptide analog, antibody or antibody fragment, metal-chelator, co-factor, aliphatic acid, carbohydrate, polynucleotides, DNA, RNA, antisense polynucleotides, carbohydrate, water-soluble dendritic, cyclodextrin, inhibition ribonucleic acid, biomaterial, nanometer Particle, spin labeling, fluorogen, the part containing metal, radioactive segment, novel functional groups and other molecule covalents or non-co- Part that group, light cage part, the actinic radiation of valency interaction can be excited, can photoisomerization part, biotin, biology Plain derivative, biotin analog, the part for being combined with heavy atom, the group chemically cracked, can photodestruciton group, extension Side chain, carbon connection sugar, redox active agent, aminothio acid, toxin part, the part through isotope marks, biology Physical probe, phosphorescence groups, chemiluminescent groups, electron dense group, magnetic group, insertion group, chromophore, energy transfer Agent, bioactivator, detectable label, small molecule, quantum dot, nanometer transmission element, radioactive nucleotides, radioactivity transmission be plain, Any combinations of neutron capture agent or above-mentioned substance, or any other required compound or material) contact.First reactive group With the second reaction-ity group reaction so that molecule is connected by [3+2] cycloaddition with alpha-non-natural amino acid.In one embodiment, First reactive group is alkynyl or azide moiety, and the second reactive group is azido or alkynyl moiety.For example, First reactive group is alkynyl moiety (including but not limited in alpha-non-natural amino acid in propargyloxyphenylalanine) And the second reactive group is azide moiety.In another example, the first reactive group be azide moiety (including (but not Be limited to) in alpha-non-natural amino acid in azido-L-phenylalanine) and the second reactive group be alkynyl moiety.
In some cases, non-naturally encoded amino acids are replaced and other additions in FGF-21 polypeptides, substitution or scarce Combination is lost to influence other biological natures of FGF-21 polypeptides.In some cases, other additions, substitution or missing can increase The stability resistance of proteolytic degradation (including but not limited to) or increase FGF-21 polypeptides of FGF-21 polypeptides to its by The compatibility of body.In some cases, other additions, substitution or missing can increase the medical stability of FGF-21 polypeptides.One In the case of a little, other additions, substitution or missing can increase the dissolubility of FGF-21 polypeptides (including but not limited in Escherichia coli Or when being expressed in other host cells).In certain embodiments, adding, replace or lacking can increase in Escherichia coli or other Polypeptide dissolubility after being expressed in recombinant host cell.In certain embodiments, in addition to the site for being incorporated to alpha-non-natural amino acid Also select another site for natural coding or alpha-non-natural amino acid substitution, this causes thin in Escherichia coli or other recombinant hosts Polypeptide dissolubility increase after being expressed in born of the same parents.In certain embodiments, FGF-21 polypeptides are comprising another addition, substitution or lack, its The compatibility to FGF-21 polypeptide receptors, associated proteins or associated ligands is adjusted, the signal after being combined with FGF-21 acceptors is adjusted Transduction, adjusts circulating half-life, regulation release or biological usability, promotes purifying or improvement or changes specific dosing way. In some embodiments, FGF-21 polypeptides are comprising increase FGF-21 variants are to the addition of the compatibility of its acceptor, substitution or lack. Similarly, FGF-21 polypeptides can include the detection (including but not limited to GFP) of improvement polypeptide, purify, pass through tissue or cell The chemistry or enzymatic lysis sequence, protease cracking of transhipment, prodrug release or the activation, the reduction of FGF-21 sizes or other characteristics of film Sequence, reactive group, antibody binding domain (including but not limited to FLAG or poly-His) or other sequences based on compatibility Row (including but not limited to FLAG, poly-His, GST etc.) or connection molecule (including but not limited to biotin).
In certain embodiments, the substitution of non-naturally encoded amino acids produces FGF-21 antagonists.In certain embodiments, Replace in relevant region is combined with acceptor or add non-naturally encoded amino acids.In certain embodiments, with heparin knot Close substitution or addition non-naturally encoded amino acids in relevant region.In certain embodiments, FGF-21 antagonists are comprising at least One makes the substitution that FGF-21 serves as antagonist.In certain embodiments, FGF-21 antagonists are included connects with water-soluble polymer The non-naturally encoded amino acids connect, it is present in the receptor binding domain of FGF-21 molecules.
In some cases, 1,2,3,4,5,6,7,8,9,10 are replaced with one or more non-naturally encoded amino acids Or 10 with upper amino acid.In some cases, FGF-21 polypeptides further comprise 1,2,3,4,5,6,7,8,9,10 or 10 Substitution with one or more non-naturally encoded amino acids to naturally occurring amino acid above.For example, in some realities Apply in example, replace one or more residues in FGF-21 with one or more non-naturally encoded amino acids.One In the case of a little, by one or more non-naturally encoded residues and the linear or branch of one or more lower molecular weights PEG connections, so that relative to the material enhancing binding affinity being connected with single higher molecular weight PEG and with can be quite Serum half-life.
In certain embodiments, there are at most two residues in FGF-21 following residue through one or more non-naturals Coded amino acid replaces.
VII. the expression in non-eukaryotic and eukaryotic
To obtain the high level expression of clone's FGF-21 polynucleotides, many of FGF-21 polypeptides of the invention will be generally encoded Nucleotides is subcloned into containing the strong promoter, transcription for instructing to transcribe/translation termination and (if with regard to the core of encoding proteins matter For acid) in the expression vector of the ribosome bind site of translation initiation.It is suitable thin known to those skilled in the art Bacterium promoter, and it is for example described in Sambrook et al. and Ausubel et al..
The bacterial expression system of FGF-21 polypeptides for expressing the present invention can be in (including but is not limited to) Escherichia coli, bud Spore bacillus (Bacillus sp.), Pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida and salmonella (Salmonella) (Palva et al., Gene 22 is obtained in：229-235(1983)；Mosbach et al., Nature302：543- 545(1983)).Kit for these expression systems is on sale on the market.Lactation known to those skilled in the art is moved The eukaryotic expression system of thing cell, yeast and insect cell, and it is also on sale on the market.Using orthogonal tRNA and aminoacyl In the case of FGF-21 polypeptides of the base tRNA synzyme (above-mentioned) to express the present invention, the host cell for expression is according to it Selected using the ability of orthogonal components.Exemplary host cell includes gram-positive bacterium (Gram-positive Bacteria) (bacillus pumilus (B.brevis), hay bacillus (B.subtilis) or streptomycete are included but is not limited to ) and gramnegative bacterium (Gram-negative bacteria) (Escherichia coli, Pseudomonas (Streptomyces) Bacterium, pseudomonas aeruginosa, pseudomonas putida), and yeast and other eukaryotics.As described herein, it can be used and include O-tRNA/O-RS pairs of cell.
The present invention eukaryotic host cell or non-eukaryotic host cell provide synthesis it is larger have consumption include non-natural ammonia The ability of the protein of base acid.On the one hand, composition optionally include (including but is not limited to) at least 10 micrograms, it is at least 50 micro- Gram, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least The protein of 10 milligrams, at least 100 milligrams, at least 1 gram or 1 gram alpha-non-natural amino acids contained above, or in vivo egg can be passed through The protein (being provided in this article on the details that recombinant protein is prepared and purified) for the amount that white matter preparation method is realized. On the other hand, the protein is optionally with (including but is not limited to) cell lysis liquid, buffer solution, medical buffer solution or other In liquid suspension (it is about about 1nl to about 100L or more than 100L to include but is not limited to volume (including but is not limited to)) (including But be not limited to) often rise to few 10 micrograms of protein, often rise to few 50 micrograms of protein, often rise to few 75 micrograms of protein, every liter At least 100 micrograms of protein, few 200 micrograms of protein are often risen to, few 250 micrograms of protein is often risen to, often rises to few 500 micrograms Protein, often rise to few 1 milligram of protein or often rise to the concentration of few 10 milligrams or more than 10 milligrams protein and be present in combination In thing.A large amount of (including but not limited to (including but not limited in vitro translated) more than with other methods is produced in eukaryotic Generally can obtainable amount) include at least one alpha-non-natural amino acid protein be the present invention feature.
The present invention eukaryotic host cell or non-eukaryotic host cell provide biosynthesis it is larger have consumption include non-day The ability of the protein of right amino acid.For example, it can be produced in cell extract, cell lysis liquid, culture medium, buffer solution etc. Raw egg white matter concentration is (including but is not limited to) at least 10 micrograms per litres, at least 50 micrograms per litres, at least 75 micrograms per litres, at least 100 Micrograms per litre, at least 200 micrograms per litres, at least 250 micrograms per litres or at least 500 micrograms per litres, at least 1 mg/litre, at least 2 milligrams/ Liter, at least 3 mg/litres, at least 4 mg/litres, at least 5 mg/litres, at least 6 mg/litres, at least 7 mg/litres, at least 8 millis G/l, at least 9 mg/litres, at least 10 mg/litres, at least 20,30,40,50,60,70,80,90,100,200,300,400, 500th, 600,700,800,900 mg/litres, 1 g/l, 5 g/l, 10 g/l or more than 10 g/l include non-natural amino The protein of acid.
I.Expression system, culture and separation
FGF-21 polypeptides can be (such as thin including yeast, insect cell, mammal in any number of suitable expression systems Born of the same parents and bacterium) middle expression.The description of exemplary expression system is provided below.
Yeast：As used herein, term " yeast " includes that the various ferment for the gene for encoding FGF-21 polypeptides can be expressed Any of mother.The yeast includes but is not limited to ascosporogenous yeast (ascosporogenous yeast) (Endomycetale (Endomycetales)), basidiospore yeast (basidiosporogenous) and Fungi Imperfecti (gemma guiding principle is belonged to (Blastomycetes)) the yeast of class.Ascosporogenous yeast is divided into Liang Ge sections：Spermophthoraceae (Spermophthoraceae) and ferment Female Cordycepps (Saccharomycetaceae).The latter includes four subfamilies, i.e. Schizosaccharomycoideae (Schizosaccharomycoideae) (for example, fission yeast (Schizosaccharomyces) belongs to), Nadsonioideae (Nadsonioideae), fat yeast subfamily (Lipomycoideae) and yeast subfamily (Saccharomycoideae) (example Such as, Pichia pastoris (Pichia) category, kluyveromyces (Kluyveromyces) category and saccharomycete (Saccharomyces) category). Basidiospore yeast includes white winter spore yeast (Leucosporidium) category, rhodosporidium toruloides (Rhodosporidium) category, lock and thrown Black powder yeast (Filobasidium) category of yeast (Sporidiobolus) category, line and incense ashes intend lock load bacterium (Filobasidiella) belong to.The yeast for belonging to Fungi Imperfecti (gemma guiding principle) class is divided into Liang Ge sections：Sporobolomycetaceae (Sporobolomycelaceae) (for example, shadow yeast (Sporoholomyces) category and cloth strangle shadow yeast (Bullera) Category) and Cryptococcaceae (Cryptococcaceae) (for example, Candida (Candida) belongs to).
Especially concerned is pichia, Kluyveromyces, Blastocystis, fragmentation for the species that the present invention is used Saccharomyces (Schizosaccharomyces), Hansenula (Hansenula), Torulopsis (Torulopsis) and vacation Species in silk saccharomyces, it includes but is not limited to pichia pastoris phaff (P.pastoris), paddy Le Shi yeast (P.guillerimondii), this primary yeast (S.carlsbergensis), saccharomyces diastaticus of saccharomyces cerevisiae, karr (S.diastaticus), Douglas yeast (S.douglasii), kluyveromyces (S.kluyveri), promise this yeast (S.norbensis), ellipsoideus yeast (S.oviformis), Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis), candida albicans bacterium (C.albicans), Candida maltosa (C.maltosa) and saccharomyces hansenii (H.polymorpha)。
The selection of yeast suitable for expressing FGF-21 polypeptides is in the range of the technical ability of those skilled in the art. When selecting the yeast host for expression, suitable host may include that display has (for example) good secretion capacity, low albumen water The yeast host of active, the good secretion capacity of solution, the generation of good soluble protein and overall robustness.Yeast can generally be obtained From a variety of sources, including but not limited to University of California's biophysics and medical physicses system yeast genes collection (Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California, Berkeley, CA) and American Type Culture collection (American Type CultureCollection, " ATCC ") (Manassas, VA).
Term " yeast host " or " yeast host cell " include can be used as or having been used as recombinant vector or other transfer DNAs Recipient yeast.After original yeast host cell of the term including having received recombinant vector or other transfer DNAs Generation.It will be appreciated that due to fortuitous mutation or intentional mutation, the offspring of single mother cell is morphologically or complementary with original parents Genomic DNA or STb gene in terms of may may not be completely the same.With that (such as will have coding FGF-21 many with relevant nature The nucleotide sequence of peptide) offspring of parent fully similar mother cell that characterizes is included in thus the referred to offspring of definition In.
The expression including extra-chromosomal replicon or integration vector and conversion carrier has been developed to be transformed into a variety of ferment In female host.For example, the expression vector for following yeast has been developed：Saccharomyces cerevisiae (Sikorski et al., GENETICS (1989)122：19；Ito et al., J.BACTERIOL.(1983)153：163；Hinnen et al., PROC.NATL.ACAD.SCI.USA (1978)75：1929)；Candida albicans bacterium (Kurtz et al., MOL.CELL.BIOL.(1986)6：142)；Candida maltosa (Kunze et al., J.BASIC MICROBIOL.(1985)25：141)；Saccharomyces hansenii (Gleeson et al., J.GEN.MICROBIOL. (1986)132：3459；Roggenkamp et al., MOL.GENETICSAND GENOMICS(1986)202：302)；Kluyveromyces fragilis (Das et al., J.BACTERIOL.(1984)158：1165)；Kluyveromyces lactis (De Louvencourt et al., J.BACTERIOL.(1983)154：737；Van denBerg et al., BIOTECHNOLOGY(NY)(1990)8：135)；Paddy Le Shi yeast (Kunze et al., J.BASICMICROBIOL.(1985)25：141)；Pichia pastoris phaff (U.S. Patent No. 5,324,639；The No. 4,929,555；With No. 4,837,148；Cregg et al., MOL.CELL.BIOL.(1985)5：3376)；Schizosaccharomyces pombe (Schizosaccharomyces pombe) (Beach et al., NATURE(1982)300：706)；And Yarrowia lipolytica (Y.lipolytica)；Aspergillus nidulans (A.nidulans) (Ballance et al., BIOCHEM.BIOPHYS.RES.COMMUN.(1983) 112：284-89；Tilburn et al., GENE(1983)26：205-221；With Yelton et al., PROC.NATL.ACAD.SCI.USA (1984)81：1470-74)；Aspergillus niger (A.niger) (Kelly and Hynes, EMBO J. (1985) 4：475-479)；Richter scale wood Mould (T.reesia) (EP 0 244 234)；And filamentous fungi, such as neurospora (Neurospora), Penicillium notatum (Penicillium), curved neck mould (Tolypocladium) (WO 91/00357), wherein each document is to be herein incorporated by reference Herein.
Those skilled in the art becomes known for the control sequence of yeast vector, and the sequence includes (but not limiting In) promoter region from following gene：For example, alcohol dehydrogenase (ADH) (EP 0 284 044)；Enolase；Glucokinase Enzyme；GPI；GAPDH (GAP or GAPDH)；Hexokinase；Phosphofructose swashs Enzyme；3-phoshoglyceric acid mutase；With pyruvate kinase (PyK) (EP 0 329 203).The yeast of encoding acid phosphatase PHO5 genes can also provide applicable promoter sequence (Miyanohara et al., PROC.NATL.ACAD.SCI.USA(1983)80： 1).Other suitable promoter sequences for yeast host may include glycerol 3-phosphate acid kinase (Hitzeman et al., J.BIOL.CHEM.(1980)255：12073) with other glycolytic enzymes (such as pyruvate decarboxylase, phosphotriose isomerase and phosphoric acid Glucose isomerase (Holland et al., BIOCHEMISTRY(1978)17：4900；Hess et al., J.ADV.ENZYME REG.(1969) 7：149) promoter).The inducible Yeast promoter of further advantage with the transcription controlled by growth conditions may include alcohol It is dehydrogenase 2, different cell pigment C, acid phosphatase, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, related to nitrogen metabolism The promoter region of the enzyme of digestive enzyme and responsible maltose and galactose utilization.Suitable for the carrier and promoter of Yeast expression It is further described in EP 0 073 657.
Yeast enhancers can be also used together with Yeast promoter.In addition, synthetic promoter also acts as Yeast promoter. For example, the upstream activating sequence (UAS) of Yeast promoter can be connected with the transcription activating region of another Yeast promoter, from And produce synthesis hybrid promoter.The example of the hybrid promoter includes the ADH regulation sequences being connected with GAP transcription activatings region Row.Referring to U.S. Patent No. 4,880, No. 734 and the 4th, 876, No. 197, it is to be incorporated herein by reference.Heterozygosis is opened Other examples of mover include connecting glycolytic enzyme gene (such as GAP by the regulatory sequence of ADH2, GAL4, GAL10 or PHO5 gene Or PyK) the promoter that is constituted of transcription activating region.Referring to EP 0,164 556.In addition, Yeast promoter may include have The naturally occurring promoter in the non-yeast source of the ability of transcription is combined and originated with yeast RNA polymerase.
May make up other control elements of a part for Yeast expression carrier includes (for example) coming from GAPDH or enolase base Terminator (Holland et al., the J.B of causeIOL.CHEM.(1981)256：1385).In addition, the duplication from 2 μ plasmid origins rises Point is applied to yeast.Suitable for Select gene trp1 genes present in yeast plasmid of yeast.Referring to Tschumper etc. People, GENE(1980)10：157；Kingsman et al., GENE(1979)7：141.Trp1 bases in tryptophan in default of growing The mutant strain of the yeast of ability provides selected marker.Similarly, Leu2 defects are supplemented by the known plasmid with Leu2 genes Type yeast strain (ATCC 20,622 or 38,626).
The known method that exogenous DNA is introduced into yeast host of those skilled in the art, and methods described is usual Including but not limited to convert spheroplast or the Whole yeast host cell handled through alkaline kation.For example, Can be according to Hsiao et al., PROC.NATL.ACAD.SCI.USA(1979)76：3829 and Van Solingen et al., J.BACT. (1977)130：Method described in 946 carries out the conversion of yeast.However, also can be such as Sambrook et al., MOLECULAR CLONING：A LAB.MANUAL(2001) it is generally described in (such as to note the DNA other methods being introduced into cell by core to use Penetrate, electroporation or protoplast fusion).Then, standard technique known to those skilled in the art can be used to cultivate ferment Female host cell.
Other methods of the known expressing heterologous albumen in yeast host cell of those skilled in the art.Generally referring to U.S. Patent Publication case the 20020055169th；U.S. Patent No. 6,361,969；No. 6,312,923；6,183,985th Number；No. 6,083,723；No. 6,017,731；No. 5,674,706；No. 5,629,203；No. 5,602,034；With No. 5,089,398；U.S.'s review patent No. RE37,343 and No. RE35,749；PCT Publication patent application case WO 99/ 07862；WO 98/37208；With WO 98/26080；European patent application EP 0 946 736；EP 0 732 403；EP 0 480 480；WO 90/10277；EP 0 340 986；EP 0 329 203；EP 0 324 274；With EP 0 164 556. Referring to Gellissen et al., ANTONIE VAN LEEUWENHOEK(1992)62(1-2)：79-93；Romanos et al., YEAST(1992) 8(6)：423-488；Goeddel, METHODS IN ENZYMOLOGY(1990)185：3-7, it is each hereby incorporated herein by In.
During the amplification stage carried out using standard feed batch fermentation process known to those skilled in the art, Yeast host bacterial strain can be made to be grown in fermentation tank.The fermentation process is applicable to explain that the carbon of specific yeast host utilizes way Footpath or the difference for expressing control model.For example, the fermentation of saccharomycete yeast host may need single glucose to feed, again Miscellaneous nitrogen source (for example, caseic hydrolysate) and multivitamin supplement.On the contrary, methylotrophic yeast pichia pastoris phaff Glycerine, methanol and trace mineral charging may be needed, but only needs simple ammonium (nitrogen) salt to carry out optimum growh and expression.Example Such as, referring to U.S. Patent No. 5,324,639；Elliott et al., J.PROTEIN CHEM.(1990)9：95；With Fieschko etc. People, BIOTECH.BIOENG.(1987)29：1113, it is to be incorporated herein by reference.
However, the fermentation process can have some common attributes unrelated with yeast host bacterial strain used.Citing comes Say, during the amplification stage, the nutrient (being usually carbon) of limiting growth can be added in fermentation tank to allow maximum growth. In addition, fermentation process usually using designed to containing appropriate carbon, nitrogen, basic salt, phosphorus and other micronutrients (vitamin, Trace mineral and salt etc.) fermentation medium.U.S. Patent No. is described in suitable for the example of the fermentation medium of Pichia pastoris In 5,324, No. 639 and the 5th, 231, No. 178, it is to be incorporated herein by reference.(it is to draw by WO2005/091944 Mode is incorporated herein) expression of the description FGF-21 in yeast.
Infect the insect cell of baculoviral：Term " insect host " or " insect host cell " refer to can be used as or used Make the insect of the recipient of recombinant vector or other transfer DNAs.The term includes the protentomon host cell transfected Offspring.It will be appreciated that due to fortuitous mutation or deliberately mutation, the offspring of single mother cell morphologically or with original parents Genomic DNA or the STb gene aspect of complementation are possible may not be identical.With that (such as will have coding FGF- with relevant nature The nucleotide sequence of 21 polypeptides) offspring of parent fully similar mother cell that characterizes is included in after thus definition is referred to Dai Zhong.
The selection of the known insect cell for being applied to expression FGF-21 polypeptides of those skilled in the art.Various insects thing Kind it is fully described in the art and on sale on the market, it includes Aedes aegypti (Aedes aegypti), silkworm (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), Spodopterafrugiperda And cabbage looper (Trichoplusia ni) (Spodopterafrugiperda).When selection is used for the insect host expressed, Suitable host may include host of the display with good secretion capacity, low proteolytic activity and overall steadiness.Insect is led to Often available from a variety of sources, including but not limited to University of California's biophysics and the insect genes preservation of medical physicses system Center (Berkeley, CA)；With American Type Culture collection (" ATCC ") (Manassas, VA).
Generally, the component of the insect expression system of infection baculoviral includes：Transfer vector (being usually bacterial plasmid), its Fragment containing Baculovirus Gene group and the facility restriction site for inserting the heterologous gene to be expressed；The shaft-like disease of wild type Poison, it has the sequence homologous with baculoviral specific fragment in transfer vector, and (this allows heterologous gene homologous recombination Into Baculovirus Gene group)；And appropriate insect host cell and growth medium.Become known for building in art Carrier, transfectional cell, the material selecting patch, make cell be grown in culture etc., methods and techniques and description can be used The handbook of these technologies.
After heterologous gene is inserted in transfer vector, carrier and wild-type virus genome are transfected into insect host In cell, wherein carrier is recombinated with viral genome.Packaged recombinant virus is expressed, and is differentiated and purified recombinant spot Block.Material and method for baculoviral/insect cell expression system can be in a kit form purchased from for example InvitrogenCorp. (Carlsbad, CA).These commonly known technologies of those skilled in the art, and the technology It is fully described in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN, the 1555th phase, in (1987), It is to be incorporated herein by reference.Referring also to RICHARDSON, 39METHODS INMOLECULAR BIOLOGY： BACULOVIRUS EXPRESSION PROTOCOLS(1995)；Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 16.9- 16.11(1994)；King and Possee, THEBACULOVIRUS SYSTEM：A LABORATORY GUIDE(1992)；And O ' Reilly etc. People, BACULOVIRUSEXPRESSION VECTORS：ALABORATORYMANUAL(1992)。
In fact, being prepared known to those skilled in the art using baculoviral/insect cell expression system various Heterologous protein.For example, referring to U.S. Patent No. 6,368,825；No. 6,342,216；No. 6,338,846；6th, No. 261,805；No. 6,245,528；No. 6,225,060；No. 6,183,987；No. 6,168,932；6,126,944th Number；No. 6,096,304；No. 6,013,433；No. 5,965,393；No. 5,939,285；No. 5,891,676；5th, No. 871,986；No. 5,861,279；No. 5,858,368；No. 5,843,733；No. 5,762,939；5,753,220th Number；No. 5,605,827；No. 5,583,023；No. 5,571,709；No. 5,516,657；No. 5,290,686；WO 02/06305；WO 01/90390；WO 01/27301；WO 01/05956；WO00/55345；WO 00/20032；WO 99/ 51721；WO 99/45130；WO 99/31257；WO 99/10515；WO 99/09193；WO 97/26332；WO 96/ 29400；WO 96/25496；WO 96/06161；WO 95/20672；WO 93/03173；WO 92/16619；WO 92/ 02628；WO 92/01801；WO 90/14428；WO 90/10078；WO 90/02566；WO 90/02186；WO 90/ 01556；WO 89/01038；WO 89/01037；WO 88/07082, it is to be incorporated herein by reference.
The known carrier for being applied to baculoviral/insect cell expression system in art, and the carrier is for example Including what is obtained from baculoviral autographa california (Autographa californica) nuclear polyhedrosis virus (AcNPV) Insect is expressed and transfer vector, and it is the virus expression carrier independent of auxiliary cell.The expressing viral obtained from this system Carrier drives the expression of heterologous gene usually using strong virus polyhedrin gene promoter.Generally referring to O ' Reilly etc. People, BACULOVIRUS EXPRESSION VECTORS：A LABORATORY MANUAL(1992)。
, generally will be (necessary comprising promoter, targeting sequencing before alien gene is inserted in shaft-like viral genome When), the said components of coded sequence of interest and transcription terminator be assembled into middle dislocation construct In (intermediatetransplacement construct) (transfer vector).Centre dislocation construct generally remains in energy Enough it is stably held in the replicon in host (such as bacterium) (such as extra-chromosomal element (for example, plasmid)).Replicon will With dubbing system, therefore keep it in the host suitable for cloning and expanding.More particularly, plasmid can contain polygonal Body protein polyadenylation signal (Miller, ANN.REV.MICROBIOL.(1988)42：177) and for being selected in Escherichia coli With protokaryon ampicillin (ampicillin) resistance (amp) gene and replication orgin of breeding.
A kind of conventional transfer vector that alien gene is introduced into AcNPV is pAc373.Also skill to art A variety of other carriers are designed known to art personnel, and the carrier includes (for example) pVL985, and it originates polyhedrin Codon changes into ATT from ATG, and it introduces BamHI cloning sites at the base-pair of 32, ATT downstreams.Referring to Luckow And Summers, VIROLOGY170：31(1989).Other commercial vectors are for example including PBlueBac4.5/V5-His； pBlueBacHis2；pMelBac；PBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
After insertion heterologous gene, by transfer vector and wild-type baculovirus genome cotransfection to insect cell place In master.The known method that allogeneic dna sequence DNA is introduced into the required site of baculoviral in art.Referring to Summers and Smith, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN, the 1555th phase, (1987)；Smith et al., MOL.CELL.BIOL.(1983)3：2156；Luckow and Summers, VIROLOGY(1989)170：31.For example, can be by same In source dual crossing restructuring insertion gene such as polyhedron gene；Also it is engineered in Baculovirus Gene needed for can be inserted into In the restriction enzyme sites crossed.Referring to Miller et al., BIOESSAYS(1989)11(4)：91.
Transfection can be realized by electroporation.Referring to TROTTERAnd WOOD, 39METHODS IN MOLECULARBIOLOGY(1995)；MANN And KING, J.GEN.VIROL.(1989)70：3501.Or, liposome can be used with recombinant expression carrier and baculovirus transfection elder brother Worm cell.For example, referring to Liebman et al., BIOTECHMQUES(1999)26(1)：36；Graves et al., BIOCHEMISTRY(1998) 37：6050；Nomura et al., J.BIOL.CHEM.(1998)273(22)：13570；Schmidt et al., PROTEIN EXPRESSION AND PURIFICATION(1998)12：323；Siffert et al., NATURE GENETICS(1998)18：45；Tilkins et al., CELL BIOLOGY：A LABORATORYHANDBOOK145-154(1998)；Cai et al., PROTEIN EXPRESSION AND PURIFICATION(1997) 10：263；Dolphin et al., NATURE GENETICS(1997)17：491；Kost et al., GENE(1997)190：139； Jakobsson et al., J.BIOL.CHEM.(1996)271：22203；Rowles et al., J.BIOL.CHEM.(L996)271(37)： 223/6；Reverey et al., J.BIOL.CHEM.(1996)271(39)：23607-10；Stanley et al., J.BIOL.CHEM. (1995)270：4121；Sisk et al., J.VIROL.(1994)68(2)：766；And Peng et al., BIOTECHNIQUES(1993)14 (2)：274.Commercially available liposomes are as included CellfectinAnd Lipofectin(Invitrogen, Corp., Carlsbad, CA).In addition, it is possible to use calcium phosphate transfection.Referring to Trotter and Wood, 39METHODS IN MOLECULAR BIOLOGY (1995)；KITTS, NAR (1990) 18 (19)：5667；And Mann and King, J.GEN.VIROL.(1989)70：3501.
Rhabdovirus expression vector usually contains bacilliform virus promoter.Bacilliform virus promoter is can be with baculoviral Any DNA sequences that RNA polymerase is combined and start code sequence (for example, structural gene) downstream (3 ') transcription enters in mRNA Row.Promoter is by with the closest transcription initiation region placed in 5 ' ends generally with coded sequence.Generally wrap this transcription initiation region Include RNA polymerase binding site and transcription initiation site.Bacilliform virus promoter can also have the second domain referred to as enhancer, When there is the domain, it is generally in the tip of structural gene.In addition, expression can be adjusted or for composition.
The structural gene largely transcribed in the latter stage of infectious cycle provides especially suitable promoter sequence.Example includes source Gene (Friesen et al., The Regulation the ofBaculovirus Gene of own coding virus multiaspect body protein Expression, THE MOLECULAR BIOLOGY OF BACULOVIRUSES(1986)；EP 0 127 839 and 0 155 476) and coding Gene (Vlak et al., the J.GEN.VIROL. (1988) 69 of p10 albumen：765) sequence.
Recently the rhabdovirus expression vector formed is packaged into infectious recombinant baculovirus and can then be passed through Technology known to those skilled in the art purifies grown patch.Referring to Miller et al., BIOESSAYS(1989)11 (4)：91；SUMMERS and Smith, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN, the 1555th phase, (1987).
Develop for infecting the recombination rhabdovirus expression vector in various insects cell.For example, developed For Aedes aegypti (ATCC the CCL-125th), silkworm (ATCC the CRL-8910th), Drosophila melanogaster (ATCC the 1963rd), Spodopterafrugiperda and the recombinant baculovirus of cabbage looper.Referring to Wright, Nature (1986) 321：718；Carbonell etc. People, J.VIROL(1985)56：153；Smith et al., MOL.CELL.BIOL.(1983)3：2156.Generally referring to Fraser et al., In Vitro CELL.DEV.BIOL.(1989)25：225.More particularly, the cell line for rod string design is usual Including but not limited to Sf9 (Spodopterafrugiperda) (ATCC the CRL-1711st), Sf21 (Spodopterafrugiperda) (Invitrogen Corp., catalog number (Cat.No.) 11497-013 (Carlsbad, CA)), Tri-368 (cabbage looper) and High-FiveTM BTI-TN-5B1- 4 (cabbage loopers).
Cell and culture medium for direct expression of the heterologous polypeptide in baculoviral/expression vector and amalgamation and expression exist It is on sale on the market, and the commonly known cell culture technology of those skilled in the art.
Escherichia coli, pseudomonad kind and other prokaryotes：Bacterial expression techniques known to those skilled in the art. Variety carrier can be used in bacterial host.Carrier can be the low or high multi-copy vector of single copy.Carrier can be used for clone and/ Or expression.In view of describing on the abundant document of carrier, the commercial availability of variety carrier and even carrier and its restriction enzyme The handbook of collection of illustrative plates and feature, herein without extensive discussions.It is well known that carrier, which is usually directed to, allows the mark of selection, These marks can provide cytotoxic agent resistance, former nutrition or immunity.Generally there are multiple marks, it provides different characteristic.
Promoters are that can combine bacterial RNA polymerase and start code sequence (such as structural gene) downstream (3 ') Any DNA sequence dna that transcription enters in mRNA.Promoter is by with the closest transcription placed in 5 ' ends generally with coded sequence Sintering.This transcription initiation region generally includes RNA polymerase binding site and transcription initiation site.Promoters can also have The second domain referred to as operator, it can be overlapping with starting the adjacent R NA polymerase binding site points of RNA synthesis.Operator Allow (inducible) transcription of negative regulation, because gene repressor protein can combine operator and thereby suppress specific gene Transcription.Can occur constructive expression in the case of in the absence of negative regulating element (such as operator).In addition, can pass through Gene activation protein binding sequence realizes positive regulation, when there is the binding sequence, and the sequence is typically closest to RNA and gathered (5 ') of synthase binding sequence.The example of gene activation protein is metabolite activated protein (CAP), and it helps to originate large intestine Transcription [Raibaud et al., the A of lac operators in bacillusNNU.REV.GENET.(1984)18：173].Therefore, adjusted expression Can be positive or negative, so as to strengthen or weaken transcription.
The sequence of encoding metabolic pathway enzyme provides especially suitable promoter sequence.Example includes deriving from glycometabolism enzyme (such as galactolipin, lactose (lac) [Chang et al., NATURE(1977)198：1056] and maltose) promoter sequence.Its Its example includes promoter sequence [Goeddel et al., the Nuc.Acids from biosynthetic enzyme (such as tryptophan (trp)) Res.(1980)8：4057；Yelverton et al., NUCL.ACIDS RES.(1981)9：731；U.S. Patent No. 4,738,921； European Published case the 036th No. 776 and the 121st No. 775, it is to be incorporated herein by reference].Beta galactosidase (bla) promoter systems [Weissmann (1981) " The cloning of interferon andother mistakes. " Interferon 3 (I.Gresser volumes)], phageλ PL [Shimatake et al., NATURE(1981)292：128] and T5 [U.S. Patent No. 4,689,406, it is to be incorporated herein by reference] promoter systems also provide applicable startup Subsequence.The preferred method of the present invention induces the FGF-21 polypeptides of high content using strong promoter (such as T7 promoters).Institute The example of these carriers known to the technical staff in category field, and it includes pET29 series and WO99/ from Novagen PPOP carriers described in 05297 (it is to be incorporated herein by reference).These expression systems produce height in host The FGF-21 polypeptides of content, without damaging host cell survival ability or growth parameter(s).PET19 (Novagen) is art In known another carrier.
In addition, non-existent synthetic promoter also functions as promoters in nature.For example, can be thin by one The transcription-activating sequence of bacterium or phage promoter is connected with the operon sequence of another bacterium or phage promoter, so as to produce GCMS computer hybrid promoter [U.S. Patent No. 4,551,433, it is to be incorporated herein by reference].For example, Tac promoters are the heterozygosis trp-lac promoters with lac operon sequences comprising trp promoters, and it is adjusted by lac repressors [Amann et al., GENE(1983)25：167；De Boer et al., PROC.NATL.ACAD.SCI.(1983)80：21].In addition, bacterium Promoter may include the naturally occurring promoter in non-bacterial source, and it, which has, is combined with bacterial RNA polymerase and originate transcription Ability.Also the naturally occurring promoter that non-bacterial can be originated is existed with compatible RNA polymerase coupling with producing some genes High level expression in prokaryotes.Phage t7 RNA polymerase/promoter systems are the examples for being coupled promoter systems [Studier et al., J.MOL.BIOL.(1986)189：113；Tabor et al., PROCNATL.ACAD.SCI.(1985)82：1074]. In addition, hybrid promoter can also include phage promoter and Escherichia coli operator region (European Published case the 267851st Number).
In addition to function on subsequence, effective ribosome bind site is also applied for expressing external base in prokaryotes Cause.In Escherichia coli, ribosome bind site is referred to as Shine-Dalgarno (SD) sequences and including initiation codon (ATG) length and at 3-11 nucleotides of upstream from start codon for 3-9 nucleotides sequence [Shine et al., Nature(1975)254：34].Think that SD sequences are matched somebody with somebody by the base between SD sequences and Escherichia coli 16S rRNA 3 ' ends To promoting mRNA and ribosomal combination [Steitz et al., " Genetic signals and Nucleotidesequences in messenger RNA ", Biological Regulation and Development： Gene Expression (Ed.R.F.Goldberger, 1979)].To express the eukaryotic gene with weak ribosome bind site With prokaryotic gene [Sambrook et al., " Expression of cloned genes in Escherichia coli ", Molecular Cloning：ALaboratory Manual, 1989].
Term " bacterial host " or " bacterial host cell " refer to can be used as or have been used as recombinant vector or other transfer DNAs Recipient bacterium.The term includes the offspring of the primitive bacteria host cell transfected.It will be appreciated that due to accidental prominent Become or deliberately mutation, the offspring of single mother cell is morphologically or in the genomic DNA complementary with original parents or STb gene side Face is possible may not be identical.With will with relevant nature (for example exist encode FGF-21 polypeptides nucleotide sequence) characterize The offspring of the abundant similar mother cell of parent is included in thus the referred to offspring of definition.
The selection of the known host bacteria for being applied to expression FGF-21 polypeptides of those skilled in the art.It is used in selection During the bacterial host of expression, suitable host may include display with good inclusion body Forming ability, low proteolytic activity with And the host of overall steadiness.Bacterial host is generally available from a variety of sources, and including but not limited to University of California gives birth to Thing physics and medical physicses system bacterial gene collection (Berkeley, CA)；And American Type Culture collection (" ATCC ") (Manassas, VA).Industry/medicine fermentation is usually using the bacterium (such as W3110) from K bacterial strains or source In the bacterium (such as BL21) of B bacterial strains.These bacterial strains are because its growth parameter(s) is known and stably particularly suitable.In addition, this A little bacterial strains are avirulence, and for security and environment reason, it is commercially more important.Suitable escherichia coli host Other examples include but is not limited to BL21, DH10B or derivatives thereof bacterial strain.In another embodiment of the inventive method In, escherichia coli host is albumen azymia bacterial strain, and it includes but is not limited to OMP- and LON-.Host cell strain can be false single Born of the same parents' strain, including but not limited to Pseudomonas fluorescens, pseudomonas aeruginosa and pseudomonas putida.Known Pseudomonas Bacterium bion 1 (being named as bacterial strain MB 101) is applied to be prepared by recombinant and available for the preparation process of therapeutic protein.It is false single The example of born of the same parents' bacterium expression system include can with host strain form be obtained from Dow Chemical Company system (Midland, MI, can be obtained by WWW on dow.com).
(that is, expression construct is introduced into host cell and isolated with conjunction forming recombinant host cell strain The host cell of suitable expression construct) after, cultivate recombinant host cell strain under conditions of suitable for generation FGF-21 polypeptides.It is affiliated The technical staff in field is it is clear that the cultural method of recombinant host cell strain is by the property dependent on the expression construct utilized The characteristics of matter and host cell.Recombinant host strain is cultivated usually using method known to those skilled in the art. Recombinant host cell be typically containing absorbable carbon, nitrogen and inorganic salts originate and optionally contain vitamin, amino Cultivated in the fluid nutrient medium of acid, growth factor and other oroteins culture supplement known to those skilled in the art. Fluid nutrient medium for cultivating host cell optionally can prevent undesirable micro- life containing antibiotic or antifungal agent The growth of thing and/or compound (antibiotic including but not limited to for selecting the host cell containing expression vector).
Recombinant host cell can pattern culture in batches or continuously, wherein cell collect (in FGF-21 polypeptides in the cell In the case of accumulation) or culture supernatants be collected as pattern in batches or continuously.Come for the preparation in prokaryotic host cell Say, preferably batch culture and cell are collected.
The FGF-21 polypeptides of the present invention are typically to be purified after being expressed in recombination system.FGF-21 polypeptides can be by affiliated Known a variety of methods are purified from host cell or culture medium in field.FGF-21 polypeptides produced by bacterial host cell There can be weak dissolubility or insoluble (being in inclusion bodies).In one embodiment of the invention, using disclosed herein Known method in method and art, can easily enter the protein that behavior increase restructuring is produced in FGF-21 polypeptides Dissolubility and the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor that selects., can be by centrifuging from host cell lysate in the case of insoluble protein Protein is collected, and can then carry out homogenizing for cell thereafter.In the case of with weak deliquescent protein, it can add Plus the including but not limited to compound of polyethyleneimine (PEI) is with the precipitation of inducing moiety soluble protein.It can then lead to Cross the albumen that precipitation is advantageously collected in centrifugation.Using a variety of methods known to those skilled in the art, recombinant host can be made Cell rupture homogenizes to discharge inclusion body from cell interior.Well known technology can be used carry out host cell rupture or Homogenize, these technologies include but is not limited to enzymatic cell rupture, sonication, Dounce and homogenized (dounce Homogenization) or high pressure release rupture.In one embodiment of the inventive method, made greatly using high pressure release tech Enterobacteria host cell ruptures to discharge the inclusion body of FGF-21 polypeptides.When handling the inclusion body of FGF-21 polypeptides, preferably make The time that homogenizes of repetitive process minimize so that inclusion body yield maximize and will not due to for example dissolving, mechanical shearing or egg The factors such as white matter hydrolysis cause damage.
Then, it can be used any of known a variety of suitable solubilizer in art insoluble or heavy to dissolve The FGF-21 polypeptides in shallow lake.Urea or guanidine hydrochloride dissolution FGF-21 polypeptides can be used.The volume of the FGF-21 polypeptides of dissolving should be made minimum Change, so as to prepare high-volume using the batch size being convenient to operation.Recombinant host can be by thousands of liters of volume batch In the large-scale commercial applications device for measuring growth, this factor can be important.In addition, working as FGF- is manufactured in large-scale commercial applications device During 21 polypeptide, for human medical's purposes, if it would be possible, should avoid that the harshness of machine and container can be damaged Chemicals or its protein.Had shown that in the method for the present invention, relatively mild denaturant urea can be used to replace harsher Denaturant guanidine hydrochloride dissolve FGF-21 polypeptide inclusion bodys.The use of urea significantly reduces manufacture of the infringement in FGF-21 polypeptides With the risk of the stainless steel equipment used in purge process, while effectively dissolving FGF-21 polypeptide inclusion bodys.
In the case of soluble FGF-21 albumen, FGF-21 can be secreted into periplasmic space or culture medium.Citing comes Say, eight difference targeting sequencings (including SEQ ID NO are included by using coding：Listed sequence in 39-44) construct Plasmid and these plasmids are transformed into W3110-B2 cells make FGF-21 be secreted into W3110-B2 cells pericentral siphon it is empty Between in, cell is grown at 37 DEG C, until OD reaches about 0.8, now with 0.01% arabinose induced expression.5 is small Shi Hou, prepares pericentral siphon release sample from culture and operates (Figure 33) on gel, overall expression (the whole bacteriolyzes of display Liquid) and Periplasmic secretion (soluble part).
In addition, solubility FGF-21 is likely to be present in the cytoplasm of host cell.It may need carrying out purification step Solubility FGF-21 is concentrated before.Standard technique known to those skilled in the art can be used from (such as) cell lysis liquid Or culture medium concentration solubility FGF-21.In addition, standard technique known to those skilled in the art can be used to rupture place Chief cell and from the cytoplasm or periplasmic space of host cell release solubility FGF-21.
When preparing the FGF-21 polypeptides in fusion protein form, fusion sequence can remove.It can be split by enzymatic or chemistry Solution realizes the removal of fusion sequence.Method known to those skilled in the art can be used to realize the enzymatic of fusion sequence Remove.It will be apparent to those skilled in the art that the selection of enzyme for removing fusion sequence will by fusion the characteristics of Determine, and reaction condition will be specified according to the selection of enzyme.Reagent (bag known to those skilled in the art can be used Include (but not limited to) cyanogen bromide, TEV protease and other reagents) realize chemical cracking.Can be by the technology people of art The known method of member purifies the FGF-21 polypeptides of cracking from the fusion sequence of cracking.Those skilled in the art will be aobvious and easy See, methods described will by fusion sequence and FGF-21 polypeptides the characteristics of and property determine.Purification process may include (but not limited to) Size exclusion chromatography, hydrophobic interaction chromatogram, ion-exchange chromatography or dialysis or its any combinations.
Also FGF-21 polypeptides can be purified to remove DNA from protein solution.Although can be by known in art Any suitable method (such as precipitation or ion-exchange chromatography) removes DNA, but also can by using nucleic acid precipitating reagent (for example (but It is not limited to) protamine sulfate) precipitate to remove DNA.The well known standard for including but not limited to centrifuging or filtering can be used Method makes FGF-21 polypeptides be separated with the DNA precipitated.By in the setting using FGF-21 polypeptides to treat the mankind, host nucleic acids The removal of molecule is key factor, and the method for the present invention drops to host cell DNA pharmaceutically acceptable level.
It is can also be used for for small-scale or large scale fermentation method in protein expression, methods described includes (but not limiting In) fermentation tank, vibration flask, fluidized bed aerosol generator, hollow-fiber bioreactor, roller bottle culture system and tank diameter Bioreactor system.Each in these methods can present material or the progress of continuous mode method in batches, in batches.
Generally, the standard method in art can be used to reclaim mankind's FGF-21 polypeptides of the present invention.For example, Culture medium or cell lysis liquid can be centrifuged or filter to remove cell fragment.Can by supernatant concentration or volume needed for being diluted to or The preparation that filter Gong is further purified into suitable buffer solution with regulation.The FGF-21 polypeptides of the present invention are further purified bag Include and FGF-21 polypeptide variants that are deamidated and cutting short form are separated from complete form.
Any of property program illustrated below can be used in the FGF-21 polypeptides of the purifying present invention：Affinity chromatography；It is cloudy from Son or cation-exchange chromatography (using (including but is not limited to) DEAE SEPHAROSE)；Silica gel chromatograph；High performance liquid chromatography (HPLC)；Reversed-phase HPLC；Gel filtration (uses (including but is not limited to) SEPHADEX G-75)；Hydrophobic interaction chromatogram； Size exclusion chromatography；Metal-chelate chromatography；Ultrafiltration/filter；Ethanol precipitation；Ammonium sulfate precipitation；Chromatofocusing；Displcement chromatography； Electrophoretic procedures (including but not limited to preparative isoelectric focusing)；Differential solubility (including but not limited to ammonium sulfate precipitation)； SDS-PAGE；Or extraction.
According to known to those skilled in the art and the standardization program that uses, can by the protein of the present invention (including (but It is not limited to) protein, the peptide comprising alpha-non-natural amino acid, the protein comprising alpha-non-natural amino acid that include alpha-non-natural amino acid Antibody, combination collocation thing etc. of the protein comprising alpha-non-natural amino acid) partially or substantially purify into homogeneous.Therefore, may be used The polypeptide of the present invention, these sides are reclaimed and purified by any of a variety of methods known to those skilled in the art Method includes but is not limited to ammonium sulfate or ethanol precipitation, acid or alkali extraction, column chromatography, affinity column chromatography, anion or cation Exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatogram, hydroxylapatite chromatography, lectin chromatogram, gel Electrophoresis etc..When preparing the mature protein correctly folded, Protein refolding steps can be used if necessary.When needing high-purity When, high performance liquid chromatography (HPLC), affinity chromatography or other suitable methods can be used in final purification step.In a reality Apply in example, purifying is used as using the antibody formed for alpha-non-natural amino acid (or protein or peptide comprising alpha-non-natural amino acid) Reagent, thus (including but is not limited to) to contain one or more alpha-non-natural amino acids protein or peptide with affine Purifying based on property.If necessary, in partial purification or after purifying into homogeneous, the polypeptide is optionally used for a variety of applications, Including but not limited to it is used as calibrating component, therapeutic agent, prophylactic, diagnosticum, investigational agent and/or exempting from as antibody generation Epidemic focus.Can be by using conventional scheme to the polypeptide of animal (preferably non-human animal) administration present invention or with epitope The antibody that fragment or cell produce to obtain the polypeptide for the present invention.Those skilled in the art can be used a variety of known Technology produces antibody.In addition, transgenic mice or other organisms (including other mammals) can be used to express people source Change antibody.Above-mentioned antibody can be used to separate or differentiate clone or the purified polypeptide of expression polypeptide.It can also be used for the present invention Polypeptide and the antibody that produces treats disease.
The polypeptide and polynucleotides of the present invention also is used as vaccine.Therefore, on the other hand, the present invention relates to one kind in lactation The method that immune response is induced in animal, it, which is included, makes mammal inoculation be enough to produce antibody and/or t cell immune response The polypeptide of the present invention of (such as T cell or cytotoxic T cell including producing cell factor) is to protect the animal against disease Disease, regardless of whether whether individual has produced the disease.Also the immune response in mammal, the side can be induced with following methods Method is included：In vivo by instructing the carrier of polynucleotides expression and coded polypeptide to transmit the polypeptide of the present invention, to induce Immune response is stated, so as to produce antibody to protect the animal against the disease of the present invention.A kind of mode of administration carrier be with The form of coating on particle makes it accelerate into required cell in other forms.The nucleic acid carrier can comprising DNA, RNA, through modification of nucleic acids or DNA/RNA heterozygotes.For as vaccine, polypeptide or nucleic acid carrier generally will be with vaccine formulation (groups Compound) form offer.Composite can further include suitable supporting agent.Because polypeptide can be decomposed under one's belt, it can be parenteral (for example, subcutaneous, intramuscular, intravenous or intracutaneous injection) administration.Composite suitable for parenteral administration includes：It is aqueous and non-aqueous Aseptic injectable solution, it containing antioxidant, buffer, bacteriostatic agent and can make isotonic molten of blood of composite and recipient Matter；And aqueous and non-aqueous sterile suspensions, it may include suspending agent or thickener.Vaccine formulation may also comprise affiliated The technical staff in field is known to be used to strengthen the adjuvant system of the immunogenicity of composite.Dosage is by regarding the given activity of vaccine Depending on and can be readily determined by normal experiment.
In addition to other bibliography indicated herein, those skilled in the art it is known that a variety of purifying/albumen Matter method for folding, it includes but is not limited to those listed in documents below：R.Scopes,Protein Purification.Springer-Verlag, N.Y. (1982)；Deutscher,Methods in Enaymology the 182nd Volume：Guide toProtein Purification, Academic Press, Inc.N.Y. (1990)；Sandana, (1997)Bioseparation ofProteins.Academic Press, Inc.；Bollag et al., (1996)Protein Methods, second edition, Wiley-Liss, NY；Walker, (1996)The Protein Protocols Handbook Humana Press, NJ；Harris and Angal, (1990)Protein Purification Applications：A Practical ApproachIRL Press at Oxford, Oxford, England；Harris and Angal,Protein Purification Methods：A Practical ApproachIRL Press atOxford, Oxford, England； Scopes, (1993)Protein Purification：Principles and Practice the 3rd editionSpringer Verlag, NY；Janson and Ryden, (1998)Protein Purification：Principles, HighResolution Methods and Applications, second edition, Wiley-VCH, NY；And Walker (1998),ProteinProtocols on CD-ROMHumana Press, NJ；And references cited therein.
The protein with alpha-non-natural amino acid of interest is produced in eukaryotic host cell or non-eukaryotic host cell Or an advantage of polypeptide is that the protein or polypeptide will generally be folded into its native conformation.However, in certain of the invention In a little embodiments, those skilled in the art should be understood that after synthesis, expression and/or purifying, and protein or peptide may With the conformation different from the conformation needed for related polypeptide.In one aspect of the invention, optionally make expressed protein or Polypeptide is denatured and then makes its renaturation.This is realized using known method in art, and methods described is included (but not Be limited to) into protein or polypeptide of interest add chaperone (chaperonin), by proteolytic in such as hydrochloric acid In the chaotropic agents such as guanidine, utilize protein disulfide-isomerase etc..
In general, it is sometimes desirable to expressed polypeptide is denatured and is reduced and then make polypeptide refolding into preferred structure As.For example, guanidine, urea, DTT, DTE and/or chaperone can be added in translation product of interest.Art Technical staff known to make protein reduction, denaturation and renaturation method (referring to above-mentioned bibliography and Debinski et al., (1993)J.Biol.Chem., 268：14065-14070；Kreitman and Pastan (1993)Bioconjug.Chem, 4： 581-585；And Buchner et al., (1992)Anal.Biochem.,205：263-270).For example, Debinski et al. is retouched State the denaturation and reduction of inclusion body protein in guanidine-DTE.(including but is not limited to) oxidized form of glutathione and L- can contained Make protein refolding in arginic redox buffer.Can make refolding reagent flow or otherwise move with One or more kinds of polypeptides or the contact of other expression products, or one or more kinds of polypeptides or other expression products can be made Flowing otherwise moves to contact with refolding reagent.
In the case where protokaryon prepares FGF-21 polypeptides, the FGF-21 polypeptides that so produce may false folding and thus Lack bioactivity or bioactivity reduction.Can be by " refolding " come the bioactivity of recoverin matter.In general, lead to Cross the one or more kinds of chaotropic agents (such as urea and/or guanidine) of use (such as) and be capable of the reducing agent (example of Reduction of Disulfide Such as dithiothreitol (DTT), DTT or 2 mercapto ethanol (2-ME)) (wherein FGF-21 polypeptides are also insoluble) is dissolved, deploys and reduces many Peptide chain and the FGF-21 polypeptide refoldings for making false folding.Under the chaotropic agent of intermediate concentration, then add oxidant (for example, Oxygen, cystine or cystamine), this allows to re-form disulfide bond.Known standard method in art can be used to make FGF-21 many Peptide refolding, methods described is for example described in U.S. Patent No. 4,511, No. 502, the 4th, 511, No. 503 and the 4th, 512, No. 922 Method, the document is to be incorporated herein by reference.FGF-21 polypeptides also can be altogether folded with shape with other oroteins Into heterodimer or heteromultimer.
After refolding, FGF-21 can be further purified.A variety of skills known to those skilled in the art can be used Art realizes FGF-21 purifying, the technology include hydrophobic interaction chromatogram, size exclusion chromatography, ion-exchange chromatography, RPLC, affinity chromatography etc. or its any combinations.Other purifying, which may also comprise, to be dried or precipitation purified protein The step of matter.
After purification, FGF-21 can be exchanged in different buffer solutions and/or by known a variety of sides in art Any of method (including but not limited to filter and dialysis) concentrates it.The FGF- provided in single protein purification form 21 can undergo aggregation and precipitate.
Purified FGF-21 can be at least 90% pure (such as by RPLC (RP-HPLC) or dodecyl sulphur Sour sodium-polyacrylamide gel electrophoresis (SDS-PAGE) is measured), or at least 95% is pure, or at least 98% pure, or at least 99% Or it is purer.The exact numerical values recited of purity regardless of FGF-21, FGF-21 is for as medical product or for further handling All it is sufficiently pure for (water-soluble polymer such as with such as PEG is engaged).
In the absence of other active components or protein (in addition to excipient, supporting agent and stabilizer, seralbumin etc.) In the case of, some FGF-21 molecules can be used as therapeutic agent, or it can be compound with another protein or polymer.
General purification process：Can be with any appropriate order to the cell lysis liquid comprising FGF-21 polypeptides, extract, culture Base, inclusion body, the periplasmic space of host cell, the cytoplasm of host cell or other materials are obtained by any separating step Any FGF-21 mixtures of polypeptides carries out any of a variety of separating steps, and any separating step includes but is not limited to It is affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatogram, gel filtration chromatography, high performance liquid chromatography (" HPLC "), anti- Phase HPLC (" RP-HPLC "), expanded bed adsorption or its any combinations and/or repetition.
For implementing the equipment and other necessary materials of technology specifically described herein on sale on the market.Pump, fraction collection Device, monitor, logger and whole system all available from (such as) Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway, NJ).Including but not limited to the chromatographic material of exchange matrix material, culture medium and buffer solution also available from These companies.
Can more quickly it be realized in balance and column chromatographic process described herein using special equipment (such as pump) Other steps (such as wash and elute).Commercially available pump includes but is not limited to HILOADPump P-50, peristaltic pump P-1, pump P- 901 and pump P-903 (Amersham Biosciences, Piscataway, NJ).The example of fraction collector includes RediFrac Fraction collector, FRAC-100 and FRAC-200 fraction collectors and SUPERFRACFraction collector (Amersham Biosciences, Piscataway, NJ).Blender can also be used to form pH value and linear concentration gradient.Commercial mixer Including gradient mixer GM-1 and pipe-line mixer (Amersham Biosciences, Piscataway, NJ).
Any commercially available monitor can be used to monitor chromatographic process.These monitors can be used for collecting such as UV, pH value and biography The information of conductance.The example of detector includes monitor UV-1, UVICORDS II, monitor UV-M II, monitor UV- 900th, monitor UPC-900, monitor pH/C-900 and conductivity monitor (Amersham Biosciences, Piscataway, NJ).In fact, whole system is all on sale on the market, it includes coming from Amersham Biosciences The various AKTA of (Piscataway, NJ)System.
In one embodiment of the invention, for example, purified FGF-21 that can be as obtained by making first in urea is more Peptide is denatured, and then dilutes to reduce FGF-21 in the TRIS buffer solutions containing reducing agent (such as DTT) under suitable pH value Polypeptide is simultaneously denatured it.In another embodiment, FGF-21 polypeptides are made to become with about 2M to about 9M concentration range in urea Property, then diluted under the pH value in the range of about 5.0 to about 8.0 in TRIS buffer solutions.This embodiment can then be cultivated Refolding mixture.In one embodiment, refolding mixture is cultivated 4 hours to 24 hours at room temperature.Then, it can enter FGF-21 mixtures of polypeptides of the one step isolated or purified through reducing and being denatured.
As described herein, before any later separation step is carried out, it can adjust the first FGF-21 mixtures of polypeptides PH value.In addition, known technology can be used in art to concentrate the first FGF-21 mixtures of polypeptides or its is any follow-up mixed Compound.In addition, can be used those skilled in the art known to technology will comprising the first FGF-21 mixtures of polypeptides or its The elution buffer of what subsequent mixtures is exchanged for the buffer solution suitable for next separating step.
Ion-exchange chromatography：In one embodiment, can be to the first FGF-21 polypeptides and as optional additional step Mixture carries out ion-exchange chromatography.Generally referring to ION EXCHANGE CHROMATOGRAPHY：PRINCIPLES ANDMETHODS(catalog number (Cat.No.) 18- 1114-21, Amersham Biosciences (Piscataway, NJ)).Commercially available ion exchange column includes HITRAP、HIPREPAnd HILOADPost (Amersham Biosciences, Piscataway, NJ).These posts utilize strong anion exchanger, example Such as Q SEPHAROSEFast Flow、Q SEPHAROSEHighPerformance and Q SEPHAROSEXL；Strong cation Exchanger, such as SP SEPHAROSEHighPerformance、SP SEPHAROSEFast Flow and SP SEPHAROSE XL；Weak anion exchanger, such as DEAE SEPHAROSEFast Flow；And weak cation exchanger, such as CM SEPHAROSEFastFlow (Amersham Biosciences, Piscataway, NJ).Can purge process any stage Anion or cation exchange column chromatography is carried out to FGF-21 polypeptides to separate substantially purified FGF-21 polypeptides.It can be used Any suitable cation exchange matrix carries out cation-exchange chromatography step.Applicable cation exchange matrix include (but It is not limited to) fibrous, porous, non-porous, particulate, bead or cross-linked cationic exchange matrix material.These cation exchange matrix Material includes but is not limited to cellulose, agarose, glucan, polyacrylate, polyethylene, polystyrene, silica, poly- The compound of ether or any of above material.
Cation exchange matrix can be any suitable cation-exchanger, including strong cation exchanger and weak cation Exchanger.Strong cation exchanger may keep ionization in wide ph range, and therefore possibility can be in wide ph range It is interior to be combined with FGF-21.However, weak cation exchanger changes such as pH value and loses and ionize.For example, pH is worked as When value drops to about pH 4 or pH below 5, weak cation exchanger may lose electric charge.Suitable strong cation exchanger includes (but not limited to) charged functional groups, such as sulfopropyl (SP), methyl sulphonyl (S) or sulfoethyl (SE).Cation exchange matrix Can be the strong cation exchanger preferably with about 2.5 to about 6.0 FGF-21 combination pH value ranges.Or, strong cation is handed over About pH 2.5 to about pH 5.5 FGF-21 combination pH value ranges can be had by changing agent.Cation exchange matrix can be with about 3.0 FGF-21 combination pH value strong cation exchanger.Or, cation exchange matrix can be for preferably with about 6.0 to about 8.0 FGF-21 combination pH value ranges strong cation exchanger.Cation exchange matrix can be for preferably with about 8.0 to about 12.5 FGF-21 combination pH value ranges strong cation exchanger.Or, strong cation exchanger can have about pH 8.0 to arrive about pH 12.0 FGF-21 combination pH value ranges.
Before FGF-21 is loaded, for example, dilute weak acid of multiple column volumes can be used (for example, the 20mM second of 4 column volumes Acid, pH 3) carry out balance cation exchange matrix.FGF-21 can be added after balance and in the substantial purified FGF-21 of elution Before, it is possible to use weak acid solution (such as weak acetic acid or phosphoric acid solution) is by post washing one to for several times.For example, it can be used about The 20mM acetic acid (pH 3) of 2-4 column volume carrys out column scrubber.For example, using the 0.05M sodium acetates (pH 5.5) of 2-4 column volume Or carry out other washings using the 0.05M sodium acetates (pH 5.5) mixed with 0.1M sodium chloride.Or, using in art The method known, dilute weak acid of multiple column volumes can be used to carry out balance cation exchange matrix.
Or, can be by making cation-exchanger matrix and the Buffer fluid contacts with sufficiently low pH value or ionic strength And replace the FGF-21 in matrix to elute substantially purified FGF-21.The pH value of elution buffer can be arrived in about pH2.5 In the range of about pH 6.0.More particularly, the pH value of elution buffer can be arrived in about pH 2.5 to about pH 5.5, about pH 2.5 In the range of about pH 5.0.Elution buffer can have about 3.0 pH value.In addition, the amount of elution buffer can be in a big way It is interior change and generally will be in the range of about 2 to about 10 column volumes.
, can be by making matrix sufficiently high with having after FGF-21 polypeptides is adsorbed onto in cation-exchanger matrix The Buffer fluid contacts of pH value or ionic strength and the FGF-21 polypeptides in matrix are replaced to elute substantially purified polypeptide.It is suitable Buffer solution for the high ph-values elution of substantially purified FGF-21 polypeptides may include (but not limited to) concentration at least about Citrate, phosphate, formates, acetate, HEPES and MES buffer solutions in the range of 5mM at least about 100mM.
Reverse-phase chromatography：Can according to known to those skilled in the art suitable scheme carry out RP-PIPLC with purifying protein Matter.Referring for example to Pearson et al., ANAL BIOCHEM.(1982)124：217-230(1982)；Rivier et al., J.CHROM. (1983)268：112-119；Kunitani et al., J.CHROM.(1986)359：391-402.RP- can be carried out to FGF-21 polypeptides HPLC is to separate substantially purified FGF-21 polypeptides.On this point, can be used contain have different lengths (including (but It is not limited to) at least about C3To at least about C30, at least about C3To at least about C20Or at least about C3To at least about C18Resin) alkyl official Can resin derived from the silica of group.Or, polymer resin can be used.For example, TosoHaas can be used Amberchrome CG1000sd resins, it is styrenic polymer resins.The cyanogen with a variety of long alkyl chains can also be used Base or polymer resin.In addition, available such as ethanol equal solvent washing RP-HPLC posts.Source RP posts are the another of RP-HPLC posts One example.
It can be used and contain ion-pairing agent and Organic modification agent (such as methanol, isopropanol, tetrahydrofuran, acetonitrile or ethanol) Suitable elution buffer from RP-HPLC posts elute FGF-21 polypeptides.The most frequently used ion-pairing agent includes but is not limited to second Acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, hyptafluorobutyric acid, triethylamine, tetramethyl-ammonium, tetrabutylammonium and acetic acid triethyl ammonium. One or more kinds of gradients or isocratic condition can be used to be eluted, wherein it is preferred that reducing disengaging time and reducing peak width Gradient condition.Another method is directed to use with two kinds of gradients with different solvents concentration range.It is applicable washing in this article Carrying the example of buffer solution may include (but not limited to) ammonium acetate and acetonitrile solution.
Hydrophobic interaction chromatogram purification technology：Hydrophobic interaction chromatogram (HIC) can be carried out to FGF-21 polypeptides. Generally referring to HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK：PRINCIPLES ANDMETHODS(catalog number (Cat.No.) 18-1020-90, Amersham Biosciences (Piscataway, NJ)), it is to be incorporated herein by reference.Suitable HIC matrix It may include the matrix that (but not limited to) replaces through alkyl or aryl, such as the matrix through the substitution of butyl, hexyl, octyl group or phenyl, The matrix includes agarose, Sepharose, Ago-Gel (sepharose), cellulose, silica, glucan, poly- Styrene, poly- (methacrylate) matrix and mixed mode resin (include but is not limited to polyethyene diamine resin or through fourth Base or poly- (methacrylate) matrix of phenyl substitution).The commercial source of hydrophobic interaction column chromatography includes (but not limiting In) HITRAP、HIPREPAnd HILOADPost (Amersham Biosciences, Piscataway, NJ).
Briefly, before loading, standard buffer solution (such as second known to those skilled in the art can be used Acid/sodium chloride solution or the HEPES containing ammonium sulfate) balance HIC posts.Ammonium sulfate can be used as loading the buffer solution of HIC posts. Load after FGF-21 polypeptides, may then use that standard buffer solution and condition carry out column scrubber to remove unwanted material, but will FGF-21 polypeptides are stayed on HIC posts.Can be (such as low containing EDTA and concentration with the standard buffer solution of about 3 to about 10 column volumes In the HEPES buffer solution of the ammonium sulfate of level pad, or acetic acid/sodium chloride buffer) elute FGF-21 polypeptides.Also it can make With for example eluting FGF-21 molecules using the linear salt gradient of the reduction of potassium phosphate gradient.Then, can be for example by filtering (example Such as filter or ultrafiltration) concentrate eluate.Filter can be used to remove the salt for eluting FGF-21 polypeptides.
Other purification techniques：First FGF-21 mixtures of polypeptides or its any subsequent mixtures can be carried out using for example solidifying Glue filters (GEL FILTRATION：PRINCIPLES AND METHODS(catalog number (Cat.No.) 18-1022-18, AmershamBiosciences, Piscataway, NJ), it is to be incorporated herein by reference), (suitable matrix is included (but not hydroxylapatite chromatography Be limited to) HA-Ultrogel, High Resolution (Calbiochem), CHT ceramic hydroxyapatites (BioRad), Bio- Gel HTP hydroxyapatites (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, filter, lyophilized etc. another separating step, with Remove any excess salt and replace the buffer solution with suitable buffer solution for next separating step or even final The allotment of drug products.
It is more to monitor FGF-21 in each step specifically described herein that technology known to those skilled in the art can be used The yield of peptide (including substantially purified FGF-21 polypeptides).The technology can also be used for being finally separating step post analysis reality The yield of purified FGF-21 polypeptides in matter.For example, multiple reversed phase high-pressures with a variety of long alkyl chains can be used Liquid-phase chromatographic column (such as cyano group RP-HPLC, C18RP-HPLC and cation exchange HPLC and gel filtration HPLC) in any Plant to monitor the yield of FGF-21 polypeptides.
In certain embodiments of the invention, FGF-21 yield can be the starting of each purification step after each purification step FGF-21 in material at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%th, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%th, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%th, at least about 98%, at least about 99%, at least about 99.9% or at least about 99.99%.
The standard technique such as SDS-PAGE can be used or is measured by using western blot method and ELISA calibratings FGF-21 polypeptides determine purity.For example, it can be directed to ferment from negative control yeast and reclaim separation with cation exchange Protein produces polyclonal antibody.The antibody can also be used for detecting the presence of contaminative host cell proteins matter.
RP-HPLC material Vydac C4 (Vydac) are made up of silica gel particle, and its surface carries C4 alkyl chains.FGF-21 is more The separation of peptide and protein impurities is the difference based on hydrophobic interaction intensity.Entered with the acetonitrile gradient in dilute trifluoroacetic acid Row elution.Preparation HPLC is carried out using stainless steel column (2.8 to 3.2 liters of Vydac C4 silica gel of filling).By adding trifluoro second Acid is acidified hydroxyapatite Ultrogel eluates and is loaded on Vydac C4 posts.Using in dilute trifluoroacetic acid Acetonitrile gradient is washed and eluted.Collect elution part and neutralized immediately with phosphate buffer.Collect in IPC limits FGF-21 polypeptide elution parts.
DEAE Sepharose (Pharmacia) materials by with the covalently bound diethyl in the surface of Sepharose beads Amino-ethyl (DEAE) group is constituted.The combination of FGF-21 polypeptides and DEAE groups is mediated by ionic interaction.Second Nitrile and trifluoroacetic acid pass through post without delay.After these materials are washed off, washed by using the acetate buffer of low ph value Post is washed to remove trace impurity.Then with neutral phosphate buffer liquid column scrubber and with have increased ionic strength buffering Liquid elutes FGF-21 polypeptides.With DEAE Sepharose fast flow packed columns.Column volume is adjusted to ensure FGF-21 polypeptides Useful load is in the range of every milliliter of gel 3-10mg FGF-21 polypeptide.Washed with water and level pad (sodium phosphate/potassium) Post.Load collecting elution part and using equilibration buffer solution post for HPLC eluates.Then use lavation buffer solution (sodium acetate Buffer solution) column scrubber, then use equilibration buffer solution.Then, with elution buffer (sodium chloride, sodium phosphate/potassium) from post Elution FGF-21 polypeptides are simultaneously collected into single elution part according to main elution overview.By washing for DEAE Sepharose posts Go out liquid regulation to specified conductivity.Gained medicine is aseptically filled into fe fluon (Teflon) bottle and is stored at -70 DEG C.
Workable other methods include but is not limited to remove endotoxic step.Endotoxin is positioned at Gram-negative Lipopolysaccharides (LPS) on the outer membrane of host cell (for example, Escherichia coli).Those skilled in the art becomes known in reduction The method of content of toxins, and methods described is including but not limited to using silica supporter, glass dust or hydroxy-apatite The purification technique of stone；Reverse-phase chromatography；Affinity chromatography；Size exclusion chromatography；Anion-exchange chromatography；Hydrophobic interaction color Spectrum；Combination of these methods etc..It may need to change or other methods are (such as common to remove pollutant from polypeptide of interest Migrate protein).Those skilled in the art becomes known for the method for measuring endotoxin content, and methods described includes (but not limited to) horseshoe crab ameboid cell lysate (Limulus Amebocyte Lysate；LAL) examine and determine.EndosafeTM- PTS is examined It is set to the barrel and the ratio of hand-held spectrophotometer using preloaded LAL reagents, chromophoric substrate and control standard endotoxin Color, single-main distribution.Alternative includes but is not limited to measure turbidity and using the dynamics LAL methods of 96 well formats.
A variety of methods and procedures can be used to carry out the FGF-21 that analysis bag contains one or more non-naturally encoded amino acids The yield and purity of protein, methods described include but is not limited to Bradford calibratings, SDS-PAGE, Silver stain SDS- PAGE, coomassie (coomassie) dyeing SDS-PAGE, mass spectrum (including but not limited to MALDI-TOF) and art The known other methods for being used to characterize protein of technical staff.
Other methods include but is not limited to：With the SDS-PAGE, Western blot, matrix of protein staining Combination of Methods Assisted laser desorption is attached/ionization massspectrum (MALDI-MS), liquid chromatography/mass spectrometry, isoelectric focusing, analytic type anion exchange, Chromatofocusing and circular dichroism spectra.
VIII. the expression in alternative system
Alpha-non-natural amino acid is introduced into non-recombinant hosts cell, mutagenesis host cell or without cell using a variety of strategies System in protein in.These systems are also applied for preparing the FGF-21 polypeptides of the present invention.Such as Lys, Cys and Tyr The derivative of amino acid with reactive side chain causes lysine to be converted into N2- acetyl-lysine.Chemical synthesis is also provided simultaneously Enter the direct method of alpha-non-natural amino acid.The newly-developed that enzymatic connection and native chemical by fragments of peptides are connected, it is possible to Manufacture is compared with larger protein.Referring for example to P.E.Dawson and S.B.H.Kent,Annu.Rev.Biochem, 69：923(2000). Chemical peptide connection and native chemical connection are described in U.S. Patent No. 6,184,344, U.S. Patent Publication case the 2004/th In No. 0138412, U.S. Patent Publication case the 2003/0208046th, WO 02/098902 and WO03/042235, the patent It is to be incorporated herein by reference.The inhibiting factor being chemically acylated using required alpha-non-natural amino acid is used The general in vitro biological synthesis method that tRNA is added in the in vitro extract that can support Protein synthesis will be super It is incorporated in the multiple proteins of substantially any size with crossing 100 alpha-non-natural amino acid locus specificities.Referring for example to V.W.Cornish, D.Mendel and P.G.Schultz,Angew.Chem.Int.Ed.Engl.1995,34：621(1995)； C.J.Noren, S.J.Anthony-Cahill, M.C.Griffith, P.G.Schultz, Ageneral method for Site-specific incorporation of unnatural amino acids into proteins,Science 244：182-188(1989)；And J.D.Bain, C.G.Glabe, T.A.Dix, A.R.Chamberlin, E.S.Diala, Biosynthetic site-specific incorporation of a unnatural amino acid into a Polypeptide,J.Am.Chem.Soc.111：8013-8014(1989).By a variety of functional groups be introduced into protein with For research protein stability, protein folding, enzyme mechanism and signal transduction.
The in vivo method being incorporated to referred to as selection pressure has been developed with using the scrambling of wild type synzyme.For example Referring to N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and R.Huber,FASEB J, 13：41(1999).The auxotrophic strain closed to the associated metabolic path that cell supplies specific natural amino acid is set to exist Grown in the minimal medium of natural amino acid containing Finite Concentration, and the transcription of target gene is suppressed.In static growth When phase starts, natural amino acid is depleted and replaced by non-natural amino acid analogs.Induction to expression of recombinant proteins makes Obtain the protein accumulation containing non-natural analogs.For example, using this tactful by adjacent fluorophenylalanine, a fluorobenzene third Propylhomoserin and P-fluoropnenylalanine are incorporated in protein, and it shows two another characteristic shoulders that can easily reflect in UV spectrum Peak, referring for example to C.Minks, R.Huber, L.Moroder and N.Budisa,Anal.Biochem.,284：29(2000)； The methionine in bacteriophage T4 Lysozyme is replaced using Trifluoromethionine, so as to pass through19F NMR study itself and chitosan oligosaccharide part Interaction, referring for example to H.Duewel, E.Daub, V.Robinson and J.F.Honek,Biochemistry,36：3404 (1997)；And have been incorporated into trifluoro leucine to replace leucine, so that the heat endurance and chemistry of leucine zipper protein Stability increase.Referring for example to Y.Tang, G.Ghirlanda, W.A.Petka, T.Nakajima, W.F.DeGrado and D.A.Tirrell, Angew.Chem.Int.Ed.Engl, 40：1494(2001).In addition, by selenomethionine and telluro egg ammonia Acid is incorporated in various recombinant proteins to promote the phased soln in X-ray crystallography.Referring for example to W.A.Hendrickson, J.R.Horton and D.M.Lemaster,EMBO J., 9：1665(1990)；J.O.Boles, K.Lewinski, M.Kunkle, J.D.Odom, B.Dunlap, LLebioda and M.Hatada,Nat.Struct.Biol., 1：283(1994)；N.Budisa, B.Steipe, P.Demange, C.Eckerskorn, J.Kellermann and R.Huber,Eur.J.Biochem., 230：788 (1995)；And N.Budisa, W.Karnbrock, S.Steinbacher, A.Humm, L.Prade, T.Neuefeind, L.Moroder and R.Huber,J.Mol.Biol.,270：616(1997).Also the egg with alkene or alkynes functional group has effectively been incorporated to Propylhomoserin analog, so as to allow to carry out other modifications to protein by chemical mode.Referring for example to J.C.M.vanHest and D.A.Tirrell,FEBS Lett., 428：68(1998)；J.C.M.van Hest, K.L.Kiick and D.A.Tirrell,J.Am.Chem.Soc.122：1282(2000)；And K.L.Kiick and D.A.Tirrell, Tetrahedron, 56：9487 (2000)；U.S. Patent No. 6,586,207；U.S. Patent Publication case the 2002/0042097th, it is by reference It is incorporated herein.
The success of this method depends on identification of the aminoacyl tRNA synthetase to non-natural amino acid analogs, the conjunction Into enzyme it is generally necessary to which high selectivity is to ensure the fidelity of protein translation.A kind of mode for the scope for extending this method is Relax the substrate specificity of aminoacyl tRNA synthetase, this reaches in the case of finite population.For example, in large intestine bar Ala is replaced by Gly in bacterium phenylalanyl tRNA synzyme (PheRS)294The size of Binding Capacity bag can be increased, and caused Fenclonine (p-Cl-Phe) is acylated tRNAPhe.Referring to, M.Ibba, P.Kast and H.Hennecke,Biochemistry, 33：7107(1994).Coli strain with this mutation PheRS allows to be incorporated to the ammonia of chlorobenzene third Acid replaces phenylalanine to bromophenyl alanine.Referring for example to M.Ibba and H.Hennecke,FEBS Lett., 364：272 (1995)；And N.Sharma, R.Furter, P.Kast and D.A.Tirrell,FEBS Lett., 467：37(2000).It is similar Ground, the point mutation Phe130Ser shown close to the amino acid binding site of Escherichia coli tyrosyl- tRNA synzyme allows nitrogen Miscellaneous tyrosine is more effectively incorporated to than tyrosine.Referring to F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soil and S.Nishimura,J.Biol.Chem., 275：40324 (2000)。
Another strategy that in vivo alpha-non-natural amino acid is incorporated in protein is conjunction of the modification with check and correction mechanism Into enzyme.These synzyme cannot be distinguished by similar with homologous natural amino acid amino acid in structure and therefore activate it. This mistake is corrected on independent site, and this makes the deacylated guarantor to keep protein translation of the mispairing amino acid from tRNA True degree.If synzyme loses check and correction activity, then the analogue of mistake activation can avoid editting function and be merged in. Confirm this method with valyl base tRNA synzyme (ValRS) at present.Referring to V.Doring, H.D.Mootz, L.A.Nangle, T.L.Hendrickson, V.de Crecy-Lagard, P.Schimmel and P.Marliere,Science, 292：501(2001).ValRS can make tRNAVal mistakes aminoacylated using Cys, Thr or aminobutyric acid (Abu)；Then, lead to Cross edit field and hydrolyze these non-homologous amino acids.After escherichia coli chromosome random mutagenesis is made, the editor in ValRS is selected There is the mutant E. coli strain of mutation in site.This editor deficiency ValRS mistakenly makes tRNAVal that Cys is housed.Cause For Abu, spatially similar with Cys (Cys-SH groups are by-the CH in Abu3Displacement), so when this mutation large intestine bar Bacteria strain in the case of there is Abu when being grown, and Abu is also incorporated in protein by mutation ValRS.Mass spectral analysis is shown in naturally About 24% valine is replaced by Abu at each valine position in protein.
Synthesis in solid state and semisynthesis have also allowed to synthesize the multiple proteins containing new amino acid.For example, Referring to following discloses case and following bibliography cited therein：Crick, F.H.C., Barrett, L.Brenner, S.Watts-Tobin, R.General nature of the genetic code for proteins.Nature, 192： 1227-1232(1961)；Hofmann, K., Bohn, H.Studies onpolypeptides.XXXVI, The effect ofpyrazole-imidazole replacements on the S-protein activating potency of an S-peptidefragment,J.Am Chem, 88 (24)：5914-5919(1966)；Kaiser, E.T.Synthetic Approaches tobiologically active peptides and proteins including enyzmes,Ace Chem Res, 22：47-54(1989)；Nakatsuka, T., Sasaki, T., Kaiser, E.T.Peptide segment Coupling catalyzed by thesemisynthetic enzyme thiosubtilisin,J Am Chem Soc, 109：3808-3810(1987)；Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides：Backbone-engineered HIV protease,Science, 256 (5054)：221-225(1992)；Chaiken, I.M.Semisynthetic peptides and Proteins,CRC Crit Rev Biochem, 11 (3)：255-301(1981)；Offord, R.E.Protein engineering by chemical meansProtein Eng., 1 (3)：151-157(1987)；And Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.A.A Designed PeptideLigasefor Total synthesis of Ribonuclease A with Unnatural Catalytic Residues,Science, 266 (5183)：243(1994).
Using chemical modification in vitro by a variety of non natural side chains including co-factor, spin labeling and oligonucleotides It is introduced into protein.Referring for example to Corey, D.R., Schultz, P.G.Generation of a hybrid sequence- Specificsingle-stranded deoxyribonuclease,Science, 238 (4832)：1401-1403(1987)； Kaiser, E.T., Lawrence D.S., Rokita, S.E.The chemical modification of enzymatic Specificity,Annu RevBiochem, 54：565-595(1985)；Kaiser, E.T., Lawrence, D.S.Chemical mutation of enyzmeactive sites,Science, 226 (4674)：505-511(1984)； Neet, K.E., Nanci A, Koshland, D.E.Properties of thiol-subtilisin, JBiol.Chem, 243 (24)：6392-6401(1968)；Polgar, L. et M.L.Bender, A new enzyme containing a synthetically formed active site.Thiol-subtilisin.J.AmChem Soc, 88：3153-3154 (1966)；And Pollack, S.J., Nakayama, G.Schultz, P.G.Introductionof nucleophiles And spectroscopic probes into antibody combining sites,Science, 242 (4881)： 1038-1040(1988)。
Or, had been used for using the aminoacyl tRNA chemically modified biological synthesis method by a variety of biophysics Probe is incorporated in the protein in vitro synthesized.Referring to following discloses case and wherein cited bibliography：Brunner, J.New Photolabeling and crosslinking methods,Annu.Rev Biochem, 62：483-514 (1993)；And Krieg, U.C., Walter, P., Hohnson, A.E.Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the Signal recognition particle,Proc.Natl.Acad.Sci, 83 (22)：8604-8608(1986).
Previously it has been shown that can pass through to the protein synthetic reaction programmed using the gene being mutated containing required amber nonsense Middle addition chemically aminoacylated inhibiting factor tRNA and in vitro by alpha-non-natural amino acid locus specificity simultaneously Enter in protein.Using these methods, the auxotrophic strain of specific amino acids can be used, is replaced with close structural homologue Several amino acids in 20 kinds of common amino acids, for example, replacing phenylalanine with fluorophenylalanine.Referring for example to Noren, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G.Ageneral method for site- Specificincorporation of unnatural amino acids into proteins,Science, 244：182- 188(1989)；M.W.Nowak et al.,Science268：439-42(1995)；Bain, J.D., Glabe, C.G., Dix, T.A., Chamberlin, A.R., Diala, E.S.Biosynthetic site-specific Incorporation of a Non-natural amino acid into apolypeptide,J.Am Chem Soc, 111：8013-8014(1989)； N.Budisa et al.,FASEB J.13：41-51(1999)；Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G.Biosyntheticmethod for introducing unnatural amino Acids site-specifically into proteins,Methods in Enz., volume 202,301-336 (1992)； And Mendel, D., Cornish, V.W. and Schultz, P.G.Site-DirectedMutagenesis with an Expanded Genetic Code,Annu Rev Biophvs.Biomol Struct.24,435-62 (1995).
For example, identification terminator codon UAG inhibiting factor tRNA is prepared and with alpha-non-natural amino acid with chemistry Mode makes its aminoacylated.Terminator codon is introduced at the site of interest in protein gene using conventional direct mutagenesis TAG.Referring for example to Sayers, J.R., Schmidt, W.Eckstein, F.5 ' -3 ' Exonuclease Inphosphorothioate-based olignoucleotide-directed mutagensis,Nucleic Acids Res, 16 (3)：791-802(1988).In vitro transcription/translation is combined to when inhibiting factor tRNA and mutator will be acylated When in system, respond UAG codons and be incorporated to alpha-non-natural amino acid, this obtains the egg for containing the amino acid in specified location White matter.Use [3H]-Phe experiment and using 'alpha '-hydroxy acids it is experimentally confirmed that in the position specified by UAG codons only It is incorporated to needed for being incorporated at any other site of amino acid and this amino acid not in protein.Referring for example to Noren et al., together Above；Kobayashi et al., (2003) Nature Structural Biology 10 (6)：425-432；And Ellman, J.A., Mendel, D., Schultz, P.G.Site-specific incorporation of novel Backbonestructures into proteins,Science, 255 (5041)：197-200(1992).
Can by any method or technology (including but not limited to chemistry or enzymatic aminoacylated) utilizes required amino acid Make tRNA aminoacylated.
Aminoacyl can be realized by aminoacyl tRNA synthetase or other enzymatic molecules (including but not limited to ribozyme) Base.Term " ribozyme " can be exchanged with " catalytic RNA ".Cech and colleague (Cech, 1987, Science, 236：1532- 1539；McCorkle et al., 1987, Concepts Biochem.64：221-226) it is confirmed the existence of and may act as the natural of catalyst There is RNA (ribozyme).Although however, only confirming that these natural RNA catalyst have cracking and montage to ribonucleic acid substrate Effect, but catalysis pedigree has been expanded to various chemical reactions by the newly-developed manually developed on ribozyme.Research has differentiated Go out to be catalyzed RNA molecule (Illangakekare et al., the 1995Science of the aminoacyl RNA keys of itself (2 ') 3 ' end 267：643-647), and amino acid can be transferred to RNA molecule (Lohse etc. of another RNA molecule from a RNA molecule People, 1996, Nature 381：442-444).
Patent Application Publication 2003/0228593 (it is to be incorporated herein by reference) description builds core The method of carbohydrase is utilizing the natural purposes encoded in making tRNA aminoacylated with non-naturally encoded amino acids with it.TRNA can be made The substrate fixed form of aminoacylated enzyme molecule (including but not limited to ribozyme) makes it possible to effectively affinity purification ammonia Acylated product.The example of suitable substrate includes agarose, Ago-Gel and magnetic beads.For aminoacylated substrate The preparation of the ribozyme of fixed form and using being described in Chemistry and Biology 2003,10：1077-1084 and In Patent Application Publication 2003/0228593, it is to be incorporated herein by reference.
The aminoacylated method of chemistry includes but is not limited to avoid during aminoacylated using synzyme by Hecht With colleague (Hecht, S.M.Ace.Chem.Res.1992,25,545；Heckler, T.G.；Roesser, J.R.；Xu, C； Chang, P.；Hecht, S.M.Biochemistry 1988,27,7254；Hecht, S.M.；Alford, B.L.；Kuroda, Y.；Kitano, S.J.Biol.Chem.1978,253,4517) and by Schultz, Chamberlin, Dougherty and its Its people (Cornish, V.W.；Mendel, D.；Schultz, P.G.Angew.Chem.Int.Ed.Engl.1995,34,621； Robertson, S.A.；Ellman, J.A.；Schultz, P.G.J.Am.Chem.Soc.1991,113,2722；Noren, C.J.；Anthony-Cahill, S.J.；Griffith, M.C；Schultz, P.G.Science 1989,244,182；Bain, J.D.；Glabe, C.G.；Dix, T.A.；Chamberlin, A.R.J.Am.Chem.Soc.1989,111,8013；Bain, J.D. Et al., Nature 1992,356,537；Gallivan, J.P.；Lester, H.A.；Dougherty, D.A.Chem.Biol.1997,4,740；Turcatti et al., J.Biol.Chem.1996,271,19991；Nowak, M.W. etc. People, Science, 1995,268,439；Saks, M.E. et al., J.Biol.Chem.1996,271,23169；Hohsaka, T. etc. People, J.Am.Chem.Soc.1999,121,34) (it is to be incorporated herein by reference) introduce method.Methods described or Other chemical aminoacylated methods can be used for making tRNA molecules aminoacylated.
The method for producing catalytic RNA can relate to produce the pond of individually random ribozyme sequences, and the pond is determined To evolution, for pond described in required aminoacylated screening active ingredients, and aminoacylated active ribozyme sequence needed for display is selected Row.
Ribozyme can include the primitive and/or region for promoting acylated activity, such as GGU primitives and the region rich in U.Citing For, the identification of amino acid substrate can be promoted by having reported the region rich in U, and GGU primitives can be formed with tRNA 3 ' ends Base-pair.The combination in GGU primitives and region rich in U is recognized while promoting amino acid with tRNA, and so as to promote tRNA 3 ' ends it is aminoacylated.
Can by using with tRNAAsn CCCGThe incomplete randomization r24mini of engagement is in vitro selected, then systemic The engineered concensus sequence found in active clone produces ribozyme.The exemplary ribozyme quilt obtained by this method Referred to as " Fx3 ribozymes " and it is described in U.S. Published Application the 2003/0228593rd (its content is by reference It is incorporated herein) in, it serves as the General Catalyst for synthesizing the various aminoacyl tRNA for being mounted with homologous non-natural amino acid.
It is fixed on substrate to can be used for facilitating aminoacylated tRNA effective compatibility to purify.The example bag of suitable substrate Include (but not limited to) agarose, Ago-Gel and magnetic beads.Ribozyme is fixed on resin using RNA chemical constitution On, for example, can be with 3 '-c/s-diol on periodate oxidation RNA ribose to produce corresponding dialdehyde, so as to promote RNA to exist Fixation on resin.Can be used various types of resins (including cheap hydrazides resin), wherein reductive amination make resin with Interaction between ribozyme produces irreversible bonded.Aminoacyl can be remarkably promoted by aminoacylated technology on this post TRNA synthesis.Kourouklis et al., Methods 2005；36：239-4 describes a kind of aminoacylated system based on post System.
It may be implemented in a variety of ways aminoacylated tRNA separation.A kind of suitable method is to be eluted with buffer solution from post Aminoacylated tRNA, sodium acetate solution of the buffer solution for example with 10mM EDTA, contains 50mM N- (2- hydroxyethyls) Piperazine-N '-(3- propane sulfonic acids), 12.5mM KCl (pH 7.0), 10mM EDTA buffer solution or the simple water through edta buffer (pH 7.0)。
Aminoacylated tRNA can be added in translation reaction the amino acid for making tRNA aminoacylated being incorporated to translation In the selected location in polypeptide produced by reaction.The example of the aminoacylated tRNA of present invention translation system, which can be used, to be included (but not limited to) cell lysis liquid.Cell lysis liquid provides the reaction group as necessary to the mRNA inputted in vitro translates polypeptide Point.The example of the reactive component includes but is not limited to ribosomal protein, rRNA, amino acid, tRNA, GTP, ATP, translated Begin and elongation factors and the other factors related to translation.In addition, translation system can be translated to translate or being spaced in batches (compartmentalized translation).Reactive component is combined in single compartment by translation system in batches, and is spaced Translation system separates translation reaction component with that can suppress the reaction product of translation efficiency.These translation systems are having on the market Sell.
In addition, coupling transcription/translation system can be used.Coupling transcription/translation system allows the DNA that will be inputted to be transcribed into Corresponding mRNA, and the mRNA is translated by reactive component.It is commercially available coupling transcription/translation example be RapidTransltion System (RTS, Roche Inc.).The system includes the mixture containing Escherichia coli lysate To provide the translation components such as ribosomes and translation factor.In addition, also include RNA polymerase with by input DNA be transcribed into MRNA templates are for translation.RTS can interleaving via reaction compartments (including supply/consumption compartment and transcription/translation compartment) The film that enters separates reactive component.
It can be carried out by other reagents (including but not limited to transferase, polymerase, catalytic antibody, multifunctional protein etc.) TRNA's is aminoacylated.
Stephan is in Scientist, on October 10th, 2005；Described in the 30-33 pages by non-naturally encoded amino acids simultaneously Enter other methods in protein.Lu et al. is in Mol Cell.2001 October；8(4)；One kind is with chemistry described in 759-69 Mode makes the method that protein is connected chemically (protein through expression is connected) with the synthetic peptide containing alpha-non-natural amino acid.
Also alpha-non-natural amino acid is incorporated in protein using microinjection.Referring for example to M.W.Nowak, P.C.Kearney, J.R.Sampson, M.E.Saks, C.G.Labarca, S.K.Silverman, W.G.Zhong, J.Thorson, J.N.Abelson, N.Davidson, P.G.Schultz, D.A.Dougherty and H.A.Lester,Science, 268：439(1995)；And D.A.Dougherty,Curr.Opin.Chem.Biol, 4：645(2000).By pawl Toad egg mother cell (Xenopus oocyte) and the following two RNA materials co-injections in vitro produced：In ammonia of interest There is the mRNA of the coding target albumen of UAG terminator codons at base acid position, and it is aminoacylated through required alpha-non-natural amino acid Amber suppressor tRNA.Then, the body translation of egg mother cell inserts alpha-non-natural amino acid the position specified by UAG Place.This method has allowed to be generally unsuitable for the in vivo structure-function research of the in vitro overall memebrane protein of expression system. Example include Fluorescent amino acid is incorporated in tachykinin neurokinin-2 receptors with measured by FRET away from From, referring for example to G.Turcatti, K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and A.Chollet,J.Biol.Chem, 271：19991(1996)；It is incorporated to biotinylation amino acid To differentiate residue that surface in ion channel exposes, referring for example to J.P.Gallivan, H.A.Lester and D.A.Dougherty,Chem.Biol, 4：739(1997)；Using the tyrasamine acid-like substance of cage with real-time monitoring ion passage In conformation change, referring for example to J.C.Miller, S.K.Silverman, P.M.England, D.A.Dougherty and H.A.Lester, Neuron, 20：619(1998)；And using α hydroxy-amino-acids to change for probing into its door control mechanism Ion channel main chain.For example, referring to P.M.England, Y.Zhang, D.A.Dougherty and H.A.Lester,Cell, 96： 89(1999)；And T.Lu, A.Y.Ting, J.Mainland, L.Y.Jan, P.G.Schultz and J.Yang,Nat.Neurosci4：239(2001).
Alpha-non-natural amino acid is directly in vivo incorporated to ability in protein a variety of advantages are provided, it is included (but not It is limited to) high yield of mutain, technology simplification, it may study in cell or in live organism the possibility of mutain Property and these mutains therapeutic treatment and diagnosis use in use.Will have various sizes, acidity, nucleophilicity, The ability that the alpha-non-natural amino acid of hydrophobicity and other properties is included in protein can greatly extend rationality and systematicness The ability of ground operon protein structure, so as to probe into protein function and produce novel protein or biology with novel property Body.
Specifically it is incorporated in para-F-Phe once trial in site, by yeast amber suppressor TRNAPheCUA/ phenylalanyl tRNA synzyme is to in p-F-Phe resistances, Phe auxotrophic E. coli bacterial strains. Referring for example to R.Furter,Protein Sci.,7：419(1998).
Also it is using the expression without acellular (in vitro) translation system acquisition FGF-21 polynucleotides of the invention It is possible.Translation system can be for cell or cell free translation system cellularity or without cell, and can be protokaryon or eucaryon Translation system.Cellular translation system includes but is not limited to wherein can be through being transcribed into mRNA and translating by required nucleotide sequence Full the cell preparation translated mRNA, such as permeation cell or cell culture.Without acellular translation system in city It is on sale on face, and it is well known that many different types and system.Example without acellular system includes (but not limiting In) prokaryotic lysate, such as Escherichia coli lysate；And eukaryotic lysate, such as malt extract, insect be thin Cell lysis bacterium solution, rabbit granulophilocyte desmacyte lysate, rabbit oocyte lysate and human cell's lysate.When gained egg White matter through glycosylation, phosphorylation or otherwise through modification when, preferable eukaryotic extract or lysate because Many modifications are only possible to occur in eukaryotic system.Some described extracts and lysate are on sale on the market (Promega；Madison, Wis.；Stratagene；La Jolla, Calif.；Amersham；Arlington Heights, Ill.；GIBCO/BRL；Grand Island, N.Y.).Also (such as dog pancreas extraction containing microsomal membrane of film extract can be used Thing), it is applied to translation secretory protein.It may include mRNA as template (in vitro translating) or including DNA as template (group Mould assembly in vitro transcription and translation) these systems in, instructed in vitro to synthesize by ribosomes.Considerable trial has been carried out to come The protein expression system that exploitation is acellular.Referring for example to Kim, D.M. and J.R.Swartz, Biotechnology and Bioengineering, 74：309-316(2001)；Kim, D.M. and J.R.Swartz, Biotechnology Letters, 22,1537-1542, (2000)；Kim, D.M. and J.R.Swartz, Biotechnology Progress, 16,385-390, (2000)；Kim, D.M. and J.R.Swartz, Biotechnology andBioengineering, 66,180-188, (1999)；And Patnaik, R. and J.R.Swartz, Biotechniques 24,862-868, (1998)；U.S. Patent No. No. 6,337,191；U.S. Patent Publication case the 2002/0081660th；WO00/55353；WO 90/05785, it is with reference Mode be incorporated herein.Another method that can be used for FGF-21 polypeptide of the expression comprising non-naturally encoded amino acids includes MRNA- peptide integration technologies.Referring for example to R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 94： 12297-12302(1997)；A.Frankel et al., Chemistry ＆Biology 10：1043-1050(2003).This In method, the mRNA templates being connected with puromycin (puromycin) are translated as peptide on ribosomes.If to a kind of or More than one tRNA molecules are modified, then also alpha-non-natural amino acid can be incorporated in peptide.Reading last mRNA After codon, puromycin captures the C-terminal of peptide.If it find that gained mRNA- peptide binding elements have in vitro examining and determine Noticeable property, then be easy to disclose its characteristic by mRNA sequence.In this way, can screen comprising one or one with The library of the FGF-21 polypeptides of upper non-naturally encoded amino acids, to differentiate the polypeptide with required property.Recently, utilization has been reported The in vitro ribosomes translation of purified components, it allows the peptide that synthesis replaces through non-naturally encoded amino acids.Referring for example to A.Forster et al., Proc.Natl Acad.Sci. (USA) 100：6353(2003).
Reconstruct translation system can also be used.Successfully using the mixture and lysate or benefit of purified translation factor Filled with purified translation factor (such as initiation factor -1 (IF-1), IF-2, IF-3 (α or β), extension factor T (EF-Tu) or whole The only factor) the combination of lysate mRNA is translated as protein.Cell free system is alternatively coupling transcription/translation system, its In as Current Protocols in Molecular Biology (F.M.Ausubel et al. edit, WileyInterscience, 1993) described in (it is specifically incorporated to herein by reference), DNA is introduced into the system In, it is transcribed into mRNA and translates the mRNA.The RNA transcribed in eukaryotic transcription system can be the ends of heteronuclear RNA (hnRNA) or 5 ' Attach the names of pre-determined candidates (7- methylguanosines) and 3 ' end plus poly A tail ripe mRNA form, it can be excellent in some translation systems Point.For example, in granulophilocyte lysate system, the mRNA attached the names of pre-determined candidates is translated with high efficiency.
IX. the macromolecule polyalcohol being coupled with FGF-21 polypeptides
Composition specifically described herein, method, technology and strategy can be used to realize to non-natural ammonia specifically described herein The various modifications of base acid polypeptide.These modifications include other functional groups being incorporated into the non-natural amino acid constituents of polypeptide, institute State functional group and include but is not limited to mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photo-crosslinking Agent；Radionuclide；Cytotoxic compound；Medicine；Affinity marker；Photoaffinity is marked；Reactive compounds；Resin； Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon hydrate Thing；Polynucleotides；DNA；RNA；Antisense polynucleotides；Carbohydrate；Water-soluble dendritic；Cyclodextrin；Inhibition ribose core Acid；Biomaterial；Nano-particle；Spin labeling；Fluorogen；Part containing metal；Radioactive segment；Novel functional groups；With it The group of its molecule covalent or noncovalent interaction；Light cage part；The part that actinic radiation can be excited；Can photoisomerization Part；Biotin；Biotin derivative；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；Can The group of photodestruciton；The side chain of extension；The sugar of carbon connection；Redox active agent；Aminothio acid；Toxin part；Through same position The part of element mark；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；Insert base Group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Quantum dot；Nanometer transmission element；Radioactive nucleus Thuja acid；Radioactivity transmission element；Neutron capture agent；Or any combinations of above-mentioned substance, or any other required compound or material. As illustrative, the non-limiting examples of composition specifically described herein, method, technology and strategy, description below will focus on Macromolecule polyalcohol is added on non-natural amino acid polypeptides, also, it is to be appreciated that related composition, method, technology and plan Slightly it is also applied for (appropriate if necessary to modify, and those skilled in the art can be carried out with the disclosure herein) addition Other functional groups (including but not limited to functional group listed above).
A variety of macromolecule polyalcohols and other molecules can be connected to adjust FGF-21 polypeptides with the FGF-21 polypeptides of the present invention Biological property and/or novel biological property is provided FGF-21 molecules.These macromolecule polyalcohols can be by naturally encoding Amino acid, any sense substituent by non-naturally encoded amino acids or natural or alpha-non-natural amino acid or be added to it is natural or Any substituent or functional group in alpha-non-natural amino acid are connected with FGF-21 polypeptides.The molecular weight of polymer can have wide model Enclose, including but not limited between about 100Da and about 100,000Da or 100, more than 000Da.The molecular weight of polymer can Between about 100Da and about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85, 000Da、80,000Da、75,000Da、70,000Da、65,000Da、60,000Da、55,000Da、50,000Da、45, 000Da、40,000Da、35,000Da、30,000Da、25,000Da、20,000Da、15,000Da、10,000Da、9,000Da、 8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3,000Da、2,000Da、1,000Da、900Da、800Da、 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymer between Between about 100Da and about 50,000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40,000Da Between.In certain embodiments, the molecular weight of polymer is between about 1,000Da and about 40,000Da.In some embodiments In, the molecular weight of polymer is between about 5,000Da and about 40,000Da.In certain embodiments, the molecular weight of polymer Between about 10,000Da and about 40,000Da.
The present invention provides the preparation of the polymer: protein conjugates of substantial homogeneous.As used herein, " substantially Homogeneous ", which is meant, observes polymer: protein conjugates molecule is more than the half of gross protein.The polymer: protein Binding element has the Pegylation FGF-21 polypeptides of bioactivity and " substantial homogeneous " of the invention provided herein Preparation is enough homogeneous so that showing the advantage of homogeneous preparation (for example, it is easy to the medicine between each batch is predicted in clinical practice Thing dynamics) preparation.
Also it may be selected to prepare polymer: the mixture of protein conjugates molecule, and advantage provided herein is It may be selected to be included in the ratio of the single polymers: protein conjugates in prime number mixture.Therefore, if necessary, it can prepare each The mixture of the polymer moieties (that is, two, three, the fourth class) being connected of kind of protein and various numbers, and by the engagement The single polymers that thing is prepared with the method using the present invention: protein conjugates are combined, and obtain with predetermined single polymerization Thing: the mixture of protein conjugates ratio.
Selected polymer can be water miscible, so that its protein connected is in aqueous environments (such as physiological environment) Do not precipitate.Polymer can be branch or non-branch.For the therapeutic use of final products preparation, polymer will be doctor It is pharmaceutically acceptable.
The example of polymer include but is not limited to poly alkyl ether and its through alkoxy end-capped analog (for example, polyoxy Ethylene glycol (polyoxyethylene glycol), polyoxyethylene glycol/propane diols and its blocked through methoxy or ethoxy Analog, especially polyoxyethylene glycol, the latter are also referred to as polyethylene glycol (polyethyleneglycol) or PEG)；Polyethylene pyrrole Pyrrolidone；Polyvinylalkylethers；Polyoxazoline, Ju Wan oxazolins and Ju Qiang base Wan oxazolins；Polyacrylamide, poly- alkane Base acrylamide and poly- hydroxy alkyl acrylamide (for example, poly- hydroxypropylmethacrylamide and its derivative)；Poly- hydroxyl Alkyl acrylate；Poly sialic acid and its analog；Hydrophilic peptide sequence；Glycan and its derivative, it includes glucan and Portugal Polysaccharid derivative, for example, Sensor Chip CM 5, dextran sulfate, aminoglucan；Cellulose and its derivative, for example, carboxylic first Base cellulose, hydroxy alkyl cellulose；Chitin and its derivative, for example, chitosan, succinyl group chitosan, carboxymethyl are several Ding Zhi, carboxymethyl chitosan；Hyaluronic acid and its derivative；Starch；Alginate；Chondroitin sulfate；Albumin；Amylopectin With carboxymethyl amylopectin；Polyaminoacid and its derivative, for example, polyglutamic acid, polylysine, poly-aspartate, poly- asparagus fern Acid amides；Copolymer-maleic anhydride, such as styrene maleic anhydride copolymer, divinyl ether copolymer-maleic anhydride；Poly- second Enol；Its copolymer；Its trimer；Its mixture；And the derivative of above-mentioned substance.
The ratio of peg molecule and protein molecule will change, and its concentration in the reactive mixture will also change. In general, available optimal ratio can be determined by the molecular weight of selected polyethylene glycol and with the number of reactive group Rate (for the efficiency for the reaction that there is the unreacted protein or polymer of minimum excess).When being related to molecular weight, generally The molecular weight of polymer is higher, and the number for the polymer molecule that can be connected with protein is fewer.Similarly, when these ginsengs of optimization During number, the branch of polymer should be taken into account.Generally, molecular weight is higher (or side chain is more), polymer: protein rate is just It is higher.
As used herein, and when covering PEG: FGF-21 polypeptide binding element, term " therapeutically effective amount " is to instigate The amount of benefit needed for patient obtains.The amount will change with individual and will include the entirety of patient depending on many factors The latent cause of disease of health and symptom to be treated.Amount for the FGF-21 polypeptides of therapy provides acceptable rate of change simultaneously And maintain required reaction on beneficial levels.Those skilled in the art's use, which can disclose the material obtained and program, to be held Change places and determine the therapeutically effective amount of the present composition.
Water-soluble polymer can be any structure type, including but not limited to linear, bifurcated or branched forms.It is water-soluble Polymer is usually poly- alkane glycol (such as polyethylene glycol (PEG)), but other water-soluble polymers can also be used.For example, Certain embodiments of the present invention is described using PEG.
PEG is well-known water-soluble polymer, and it is on sale on the market or can be according to those skilled in the art Known method (Sandler and Karo, Polymer Synthesis, Academic Press, New York, volume 3, 138-161 pages) prepared by the ring-opening polymerisation of ethylene glycol.Term " PEG " be widely used in cover any peg molecule and Without considering PEG size or the modification of its end, and it can be expressed from the next to be connected with FGF-21 polypeptides：
XO-(CH2CH2O)n-CH2CH2- Y,
Wherein n is that 2 to 10,000 and X is H or end modified, and it includes but is not limited to C1-4Alkyl, protection group or end End functional group.
In some cases, PEG used in the present invention is at one end with hydroxyl or methoxy group, i.e. X is H or CH3 (" methoxyl group PEG ").Or, PEG can be blocked with reactive group, so as to form double functional copolymer.Typical reaction group It may include to be generally used for (to include but is not limited to horse with the reactive group of the functional group reactionses seen in 20 kinds of common amino acids Come imide, activated carbonate (including but not limited to p-nitrophenyl ester), Acibenzolar and (include but is not limited to N- hydroxyl ambers Amber acid imide, p-nitrophenyl ester) and aldehyde) and it is inert but with being deposited in non-naturally encoded amino acids to 20 kinds of common amino acids Complementary functional groups specific reaction functional group (including but not limited to azido, alkynyl).It should be noted that PEG's is another End (being represented in above formula by Y) will be directly connected to or indirect by naturally occurring or non-naturally encoded amino acids with FGF-21 polypeptides It is connected with FGF-21 polypeptides.For example, Y can be the amido (the including but not limited to ε amine or N-terminal of lysine) with polypeptide The acid amides of connection is bonded, carbamate is bonded or urea is bonded.Or, Y can be and thiol group (including but not limited to half The thiol group of cystine) connection maleimide it is bonded.Or, Y can be with generally can not by 20 kinds of common amino acids It is bonded that close residue is connected.For example, the azido on PEG can react to be formed not with the alkynyl on FGF-21 polypeptides This root [3+2] cycloaddition product.Or, alkynyl on PEG can be reacted with azido present in non-naturally encoded amino acids with Form similar product.In certain embodiments, strong nucleopilic reagent (including but not limited to hydrazine, hydrazides, azanol, semicarbazides) can be with The reaction of aldehydes or ketones base present in non-naturally encoded amino acids is to form hydrazone, oxime or semicarbazones, in some cases, when appropriate Hydrazone, oxime or semicarbazones can be further reduced by being handled by appropriate reducing agent.Or, strong nucleopilic reagent can pass through non-natural Coded amino acid is incorporated in FGF-21 polypeptides and can be used for preferentially reacting with ketone present in water-soluble polymer or aldehyde radical.
Can optionally it include but is not limited to according to the PEG being actually needed using any molecular mass, the molecular mass About 100 dalton (Dalton；Da (including but not limited to it is) 0.1- sometimes to 100,000Da or more than 100,000Da 50kDa or 10-40kDa).PEG molecular weight can have wide scope, including but not limited between about 100Da and about 100, Between 000Da or more than 100,000Da.PEG can between about 100Da and about 100,000Da, including but not limited to 100, 000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60, 000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In certain embodiments, PEG is between about 100Da and about 50,000Da.In certain embodiments, PEG between about 100Da with Between about 40,000Da.In certain embodiments, PEG is between about 1,000Da and about 40,000Da.In some embodiments In, PEG is between about 5,000Da and about 40,000Da.In certain embodiments, PEG is between about 10,000Da and about 40, Between 000Da.Side chain PEG can also be used, it, which includes but is not limited to each chain, has in 1-100kDa (including but not limited to 1- 50kDa or 5-20kDa) in the range of MW PEG molecules.The molecular weight of side chain PEG each chain (can include but is not limited to) between Between about 1,000Da and about 100,000Da or 100,000Da.The molecular weight of side chain PEG each chain can between about 1,000Da with Between about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da、70,000Da、65,000Da、60,000Da、55,000Da、50,000Da、45,000Da、40,000Da、35, 000Da、30,000Da、25,000Da、20,000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6, 000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1,000Da.In certain embodiments, side chain PEG each chain Molecular weight is between about 1,000Da and about 50,000Da.In certain embodiments, the molecular weight of side chain PEG each chain between Between about 1,000Da and about 40,000Da.In certain embodiments, the molecular weight of side chain PEG each chain between about 5,000Da with Between about 40,000Da.In certain embodiments, the molecular weight of side chain PEG each chain is between about 5,000Da and about 20,000Da Between.A variety of PEG molecules are described in and (included but is not limited to) Shearwater Polymers, Inc. catalogue, Nektar In Therapeutics catalogues, the catalogue is incorporated herein by reference.
Generally, at least one end of PEG molecules can be used for reacting with non-naturally encoded amino acids.For example, it can make Make PEG and non-day as described herein with the PEG derivatives with the alkynyl and azide moiety reacted with amino acid side chain Right coded amino acid connection.If non-naturally encoded amino acids include azido, then PEG will generally be realized containing alkynyl moiety The formation of [3+2] cycloaddition product, or the activation PEG materials (that is, ester, carbonic ester) containing phosphino- are to realize that amido link is formed. Or, if non-naturally encoded amino acids include alkynes, then PEG, which will generally contain, to be realized azide moiety to realize [3+2] Hughes The formation of root cycloaddition product.If non-naturally encoded amino acids include carbonyl, then PEG generally will include effective nucleophilic respectively Reagent (including but not limited to hydrazides, hydrazine, azanol or semicarbazide function) is to realize the shape of corresponding hydrazone, oxime and semicarbazones key Into.In other alternative solutions, the opposite orientation of above-mentioned reactive group can be used, i.e. can make in non-naturally encoded amino acids Azide moiety and the PEG derivatives reactions containing alkynes.
In certain embodiments, contain can be with non-naturally encoded amino for the FGF-21 polypeptide variants with PEG derivatives The chemical functional group that chemical functional group present on sour side chain reacts.
In certain embodiments, the present invention provides the polymer derivant containing azido and acetylene, and it, which is included, has about Water-soluble polymer main chains of the 800Da to about 100,000Da mean molecule quantity.The main polymer chain of water-soluble polymer can be Polyethylene glycol.It should be appreciated, however, that including but not limited to polyethylene glycol and other related polymers (including poly- glucan and poly- third Glycol) a variety of water-soluble polymers be also applied for implement the present invention, and term PEG or polyethylene glycol use intend cover And including all these molecules.Term PEG including but not limited to be in any type of polyethylene glycol, including difunctionality PEG, Multi-arm PEG, derivative PEG, bifurcated PEG, side chain PEG, side joint PEG are (i.e. with one or more side joints to main polymer chain On functional group PEG or related polymer) or with degradable bonded PEG.
PEG is generally transparent, colourless, tasteless, it is water-soluble in, it is inert to many chemical reagent to thermally-stabilised, no Hydrolysis is rotten, and generally nontoxic.Think polyethylene glycol for biocompatibility, that is to say, that PEG can with living tissue or Organism is coexisted without causing harm.More particularly, PEG is essentially non-immunogenic, that is to say, that PEG is not inclined to Immune response is produced in vivo.When with having some to want the molecule (such as bioactivator) of function to be connected in vivo, PEG inclines To in sheltering the reagent and any immune response can be reduced or eliminated, so that organism can tolerate depositing for the reagent .PEG binding elements tend to not produce substantive immune response or cause blood coagulation or other effects being not required to.With formula-CH2CH2O- (CH2CH2O)n-CH2CH2- (wherein n be about 3 to about 4000, normally about 20 to PEG about 2000) be applied to it is of the invention.At this In some embodiments of invention, the PEG with about 800Da to about 100,000Da molecular weight is particularly suitable as polymerizeing owner Chain.PEG molecular weight can have wide scope, including but not limited between about 100Da and about 100,000Da or 100,000Da Between above.PEG molecular weight can between about 100Da and about 100,000Da, including but not limited to 100,000Da, 95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60,000Da、55, 000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20,000Da、15, 000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3,000Da、2, 000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In some realities Apply in example, PEG molecular weight is between about 100Da and about 50,000Da.In certain embodiments, PEG molecular weight between Between about 100Da and about 40,000Da.In certain embodiments, PEG molecular weight between about 1,000Da and about 40,000Da it Between.In certain embodiments, PEG molecular weight is between about 5,000Da and about 40,000Da.In certain embodiments, PEG Molecular weight between about 10,000Da and about 40,000Da.
Main polymer chain can be linear or branch.Commonly known branched polymers main chain in art.Generally, branch Polymer has center branch core and multiple linear polymer chains being connected with center branch core.PEG is usual Used with branched forms, the form can by by ethylene oxide be added to various polyalcohols (for example glycerine, glycerine oligomer, Pentaerythrite and D-sorbite) on prepare.Center branch part point also may originate from several amino acids (such as lysine).Branch Polyethylene glycol can R (- PEG-OH) in general formmRepresent, wherein R is derived from such as glycerine, glycerine oligomer or pentaerythrite Deng core, and m represents the number of arm.Multi-arm PEG molecules are (such as in U.S. Patent No. 5,932,462；5,643rd, No. 575；No. 5,229,490；No. 4,289,872；U.S. patent application case 2003/0143596；WO 96/21469；And Multi-arm PEG molecules described in WO 93/21259, the document is each incorporated herein in entirety by reference) also it can use Make main polymer chain.
Branch PEG is alternatively by PEG (- YCHZ2)nThe bifurcated PEG forms of expression, wherein Y is linking group and Z is logical The activated terminus group that the atomic link and CH for crossing specific length are connected.
Another branched forms side joint PEG has the reaction such as carboxyl along PEG main chains rather than in the end of PEG chains Property group.
In addition to these PEG forms, can also prepare has low binding or degradable bonded polymer in main chain.Citing comes Say, the PEG in main polymer chain with the ester bond connection for being subjected to hydrolysis can be prepared.As shown below, this hydrolysis makes Polymer cracking is the fragment of lower molecular weight：
-PEG-CO2-PEG-+H2O→PEG-CO2H+HO-PEG-。
It will be understood by one of ordinary skill in the art that term polyethylene glycol or PEG are represented or including known in art Form of ownership, it includes but is not limited to form disclosed herein.
Many other polymer are also applied in the present invention.In certain embodiments, the water with 2 to about 300 ends Soluble polymer main chain is particularly suitable for use in the present invention.The example of suitable polymer includes but is not limited to other poly- alkane two Alcohol, such as polypropylene glycol (" PPG "), its copolymer (the including but not limited to copolymer of ethylene glycol and propane diols), its trimerization Thing, its mixture etc..Although the molecular weight of each chain of main polymer chain can change, its generally in about 800Da to about 100, 000Da, normally about 6,000Da are in the range of about 80,000Da.The molecular weight of each chain of main polymer chain can be between about 100Da Between about 100,000Da, including but not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80, 000Da、75,000Da、70,000Da、65,000Da、60,000Da、55,000Da、50,000Da、45,000Da、40, 000Da、35,000Da、30,000Da、25,000Da、20,000Da、15,000Da、10,000Da、9,000Da、8,000Da、 7,000Da、6,000Da、5,000Da、4,000Da、3,000Da、2,000Da、1,000Da、900Da、800Da、700Da、 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of each chain of main polymer chain Between about 100Da and about 50,000Da.In certain embodiments, the molecular weight of each chain of main polymer chain is between about Between 100Da and about 40,000Da.In certain embodiments, the molecular weight of each chain of main polymer chain between about 1,000Da with Between about 40,000Da.In certain embodiments, the molecular weight of each chain of main polymer chain is between about 5,000Da and about 40, Between 000Da.In certain embodiments, the molecular weight of each chain of main polymer chain between about 10,000Da and about 40,000Da it Between.
Those skilled in the art should be understood that the above-mentioned inventory of substantially water-soluble main chain is anyway and not exhaustive And only have illustrative, and expected all polymeric materials with above-mentioned property are suitable in the present invention.
In some embodiments of the invention, polymer derivant is " polyfunctional ", means that main polymer chain has At least two ends through functional group's functionalization or activation and there may be up to about 300 ends.Multifunctional polymer spreads out Linear polymer of the biology including but not limited to two ends, wherein each end and the functional group's key that may be the same or different Knot.
In one embodiment, polymer derivant has following structure：
X-A-POLY-B-N=N=N,
Wherein：
N=N=N is azide moiety；
B is coupling part, and it may be present or is not present；
POLY is water-soluble non-antigenic polymers；
A is coupling part, and it may be present or is not present and it can be identical or different with B；And
X is second functional group.
The example of A and B coupling part including but not limited to contains up to 18 and can be containing 1-10 carbon atom Multifunctional alkyl.It may include the hetero atom such as nitrogen, oxygen or sulphur in alkyl chain.Alkyl chain also can punish branch in hetero atom.A and Other examples of B coupling part include but is not limited to containing up to 10 and can be containing the multifunctional of 5-6 carbon atom Aryl.One or more carbon atoms of aryl can replace through nitrogen, oxygen or sulphur atom.Other examples of suitable linking group Including U.S. Patent No. 5,932,462；No. 5,643,575；And in Patent Application Publication 2003/0143596 Described linking group, the document is each incorporated herein by reference.Those skilled in the art should be understood that The above-mentioned inventory of coupling part is anyway and not exhaustive and only have illustrative and expected all with above-mentioned property Coupling part is suitable in the present invention.
The example for being suitable for X functional group includes but is not limited to hydroxyl, through protecting hydroxyl, alkoxy, active ester (for example N-hydroxy-succinamide ester and 1- BTAs ester), activated carbon acid esters (such as carbonic acid N-hydroxy-succinamide ester and carbonic acid 1- BTAs ester), acetal, aldehyde, aldehydrol, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, Aminooxy group, through protect amine, hydrazides, through protect hydrazides, through protect mercaptan, carboxylic acid, through protect carboxylic acid, isocyanates, isothiocyanic acid Ester, maleimide, vinyl sulfone, dithiopyridines, vinylpyridine, iodoacetamide, epoxides, glyoxal, diketone, first Sulphonic acid ester, tosylate, trifluoro esilate, alkene, ketone and azide.It will be understood by one of ordinary skill in the art that institute Select X section compatible with azido with so that not reacted with azido.Polymer derivant containing azido can be with double Function, mean that second functional group (that is, X) is also azide moiety；Or be Heterobifunctional, mean second functional group for not Same functional group.
Term " through protection " refers to there is the guarantor for preventing chemical reactivity functional group from being reacted under some reaction conditions Protect base or protection part.Protection group should be depending on being protected chemically reactive group type and change.For example, if chemical Reactive group is amine or hydrazides, then protection group may be selected from tertbutyloxycarbonyl (t-Boc) and 9- fluorenylmethoxycarbonyl groups (Fmoc) group.If chemically reactive group is mercaptan, then protection group can be adjacent pyridyl disulfide.If chemical Reactive group is carboxylic acid (such as butyric acid or propionic acid) or hydroxyl, then protection group can be benzyl or such as methyl, ethyl or uncle The alkyl such as butyl.Known other protection groups can also be used in the present invention in art.
The particular instance of functional end-group includes but is not limited to carbonic acid N- succinimide esters (referring for example to U.S. in document State's patent the 5th, 281, No. 698, the 5th, 468, No. 478), amine is (referring for example to Buckmann et al., Makromol.Chem.182： 1379(1981)；Zalipsky et al., Eur.Polym.J.19：1177 (1983)), hydrazides (referring for example to Andresz et al., Makromol.Chem.179：301 (1978)), succinimidyl propionate and butyric acid succinimide ester be (referring for example to Olson Et al., Poly (ethylene glycol) Chemistry ＆ Biological Applications, the 170-181 pages, Harris and Zalipsky is compiled, ACS, Washington, D.C., 1997；Referring also to U.S. Patent No. 5,672,662), amber Sour succinimide ester is (referring for example to Abuchowski et al., Cancer Biochem.Biophys.7：175 (1984) and Joppich et al., Makromol.Chem.180：1381 (1979)), succinimide ester (referring for example to U.S. Patent No. 4, No. 670,417), carbonic acid BTA ester (referring for example to U.S. Patent No. 5,650,234), glycidol ether (referring for example to Pitha et al., Eur.J Biochem.94：11(1979)；Elling et al., Biotech.Appl.Biochem.13：354 (1991)), oxygen carbonyl imidazoles is (referring for example to Beauchamp et al., Anal.Biochem.131：25(1983)；Tondelli etc. People, J.Controlled Release 1：251 (1985)), p-nitrophenyl carbonate ester (referring for example to Veronese et al., Appl.Biochem.Biotech., 11：141(1985)；And Sartore et al., Appl.Biochem.Biotech., 27： 45 (1991)), aldehyde is (referring for example to Harris et al., J.Polym.Sci.Chem.Ed.22：341(1984)；U.S. Patent No. 5, 824, No. 784, U.S. Patent No. 5,252,714), maleimide is (referring for example to Goodson et al., Biotechnology (NY)8：343(1990)；Romani et al., Chemistry of Peptides and Proteins 2：29(1984))；With And Kogan, Synthetic Comm.22：2417 (1992)), adjacent pyridyl disulfide (referring for example to Woghiren et al., Bioconj.Chem.4：314 (1993)), propenyl is (referring for example to Sawhney et al., Macromolecules, 26：581 (1993)), vinyl sulfone (referring for example to U.S. Patent No. 5,900,461).All above-mentioned bibliography and patent be all with The mode of reference is incorporated herein.
In certain embodiments of the present invention, polymer derivant of the invention includes the polymerization owner with following structure Chain：
X-CH2CH2O-(CH2CH2O)n-CH2CH2- N=N=N,
Wherein：
X is functional group as described above；And
N is about 20 to about 4000.
In another embodiment, polymer derivant of the invention includes the main polymer chain with following structure：
X-CH2CH2O-(CH2CH2O)n-CH2CH2-O-(CH2)m- W-N=N=N,
Wherein：
W is the aliphatic comprising 1-10 carbon atom or aromatic series coupling part；
N is about 20 to about 4000；And
X is functional group as described above, and m is between 1 and 10.
The present invention can be prepared by a variety of methods known in art and/or disclosed herein contains azido PEG derivatives.In a kind of method shown below, the mean molecule quantity with about 800Da to about 100,000Da it is water-soluble Property main polymer chain (main polymer chain have with the first functional group be bonded first end and with suitable leaving group be bonded Second end) with azido anion (its can with a variety of suitable counter ion counterionsl gegenions (including sodium, potassium, tert-butyl group ammonium etc.) Any pairing) reaction.Leaving group undergoes nucleophilic displacement and is replaced by azide moiety, thus needed for obtaining containing azido PEG polymer.
X-PEG-L+N3 -→X-PEG-N3。
It is as implied above, it is adaptable to which that there is main polymer chain of the invention Formula X-PEG-L, wherein PEG to be polyethylene glycol and X For not with azido react functional group, and L be suitable leaving group.The example of suitable functional group includes but is not limited to Hydroxyl, through protect hydroxyl, acetal, alkenyl, amine, aminooxy group, through protect amine, through protect hydrazides, through protect mercaptan, carboxylic acid, through protect Protect carboxylic acid, maleimide, dithiopyridines and vinylpyridine and ketone.The example of suitable leaving group includes (but not limiting In) chlorion, bromide ion, iodide ion, methanesulfonate, trifluoro ethyl sulfonic acid root and tosylate.
In the method for another polymer derivant containing azido for preparing the present invention, make with azido functional group Bridging agent with about 800Da to about 100,000Da mean molecule quantity water-soluble polymer main chain contact, wherein connecting Agent has the chemical functional group with chemical functional group's selective reaction on PEG polymer, to form the polymerization containing azido Thing derivative products, wherein azido are to be separated by linking group with main polymer chain.
Exemplary reaction process is presented below：
X-PEG-M+N- connexons-N=N=N → PG-X-PEG- connexon-N=N=N,
Wherein：
PEG is polyethylene glycol and X is the end-capping group such as alkoxy or functional group as described above；And
M for can not with azido functional group reactionses but can with N functional groups effectively and selective reaction functional group.
The example of suitable functional group includes but is not limited to：If N is amine, then M is carboxylic acid, carbonic ester or activity Ester；If N is hydrazides or aminoxyl moiety, then M is ketone；If N is nucleophilic group, then M is leaving group.
The pure of crude product can be realized by known method (including but not limited to precipitated product, then carries out chromatogram if necessary) Change.
The particularly example in the case of PEG diamines is set out below, one of amine such as the tert-butyl group-Boc through protecting Protect base section protection and the mono-protected PEG diamines of gained reacts with the coupling part with azido functional group：
BocHN-PEG-NH2+HO2C-(CH2)3- N=N=N.
In this case, a variety of activators (such as thionyl chloride or carbodiimide reagent and N- hydroxyls can be used Succinimide or N- hydroxybenzotriazoles) amido is coupled with hydroxy-acid group with monoamine PEG derivatives and with azido Coupling part between produce amido link.After amido link is successfully formed, gained contains nitrine through what the N- tert-butyl groups-Boc- was protected The derivative of base can be directly used for modified biological bioactive molecule, or it further can install other applicable officials through fine modification Can group.For example, the hydrolysis of N-t-Boc groups can be made to produce omega-amino-PEG- azide by using strong acid treatment.Can Gained amine is used as synthesis handle (synthetic handle) other applicable functional group's (such as maleimides are installed Group, activated disulfide, Acibenzolar etc.) to produce valuable Heterobifunctional reagent.
When needing each end connection by different molecular and polymer, Heterobifunctional derivative is particularly useful.Citing comes Say, ω-N- amino-N- azidos PEG will allow active electrophilic group (such as aldehyde, ketone, active ester, activated carbon acid esters Deng) molecule and a PEG end connect, and molecule with acetylene group and PEG another end will be allowed to connect Connect.
In another embodiment of the present invention, polymer derivant has following structure：
X-A-POLY-B-C ≡ C-R,
Wherein：
R can be H or alkyl, alkene, alkoxy or aryl or substituted aryl；
B is coupling part, and it may be present or is not present；
POLY is water-soluble non-antigenic polymers；
A is coupling part, and it may be present or is not present and it can be identical or different with B；And
X is second functional group.
The example of A and B coupling part includes but is not limited to containing up to 18 and can be containing 1-10 carbon atom Multifunctional alkyl.It may include the hetero atom such as nitrogen, oxygen or sulphur in alkyl chain.Alkyl chain also can punish branch in hetero atom.A and B Other examples of coupling part including but not limited to contain up to 10 and can the multifunctional virtue containing 5-6 carbon atom Base.One or more carbon atom atoms of aryl can replace through nitrogen, oxygen or sulphur atom.Suitably linking group is other Example includes U.S. Patent No. 5,932,462 and No. 5,643,575 and Patent Application Publication 2003/ Linking group described in 0143596, the document is each incorporated herein by reference.Those skilled in the art It should be understood that the above-mentioned inventory of coupling part anyway and it is not exhaustive and only have it is illustrative, and it is expected have it is above-mentioned A variety of coupling parts of property are suitable for the present invention.
Being suitable for the example of X functional group includes hydroxyl, through protecting hydroxyl, alkoxy, active ester (such as N- hydroxysuccinimidyls Imide ester and 1- BTAs ester), activated carbon acid esters (such as carbonic acid N-hydroxy-succinamide ester and carbonic acid 1- BTAs Ester), acetal, aldehyde, aldehydrol, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, aminooxy group, warp Protect amine, hydrazides, through protecting hydrazides, through protecting mercaptan, carboxylic acid, through protecting carboxylic acid, isocyanates, isothiocyanates, Malaysia acyl Imines, vinyl sulfone, dithiopyridines, vinylpyridine, iodoacetamide, epoxides, glyoxal, diketone, methanesulfonates, first Benzene sulfonate and trifluoro esilate, alkene, ketone and acetylene.It will be appreciated that selected X section should be compatible with acetenyl to cause not Reacted with acetenyl.Polymer derivant containing acetylene can be, with difunctionality, to mean second functional group (that is, X) It is acetylene moiety, or Heterobifunctional, it is different functional groups to mean second functional group.
In another embodiment of the present invention, polymer derivant includes the main polymer chain with following structure：
X-CH2CH2O-(CH2CH2O)n-CH2CH2-O-(CH2)m- C ≡ CH,
Wherein：
X is functional group as described above；
N is about 20 to about 4000；And
M is between 1 and 10.
The particular instance of each Heterobifunctional PEG polymer is presented below.
It can be used those skilled in the art known and/or method disclosed herein contain second prepare the present invention The PEG derivatives of alkynes.In one approach, the water-soluble polymeric to about 100,000Da mean molecule quantity with about 800Da is made (main polymer chain has the first end being bonded with the first functional group and be bonded with suitable nucleophilic group the to owner's chain Two ends) with being reacted with acetylene functional groups and suitable for the compound with the leaving group of the nucleophilic group reaction on PEG.When with The PEG polymer of nucleophilic moiety is with having during the molecular combinations of leaving group, and leaving group undergoes nucleophilic displacement and by nucleophilic moiety Displacement, so that the polymer containing acetylene needed for obtaining.
X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′。
As it appears from the above, for the preferred polymers main chain in reaction there is Formula X-PEG-Nu, wherein PEG to be polyethylene glycol, Nu is nucleophilic moiety and X is the functional group do not reacted with Nu, L or acetylene functional groups.
Nu example includes but is not limited to the main amine reacted by SN2 types mechanism, alkoxy, aryloxy group, sulfydryl, Asia Amino, carboxylic acid ester groups, hydrazide group, aminooxy group.Other examples of Nu groups include what is mainly reacted by nucleophilic addition Functional group.The example of L groups includes chlorion, bromide ion, iodide ion, methanesulfonate, trifluoro ethyl sulfonic acid root and tosylate With other groups of expected experience nucleophilic displacement, and ketone, aldehyde, thioesters, alkene, alpha-beta unsaturated carbonyl, carbonate group and expection Undergo other electrophilic groups of nucleopilic reagent addition.
In another embodiment of the present invention, A is aliphatic linkers with 1-10 carbon atom or with 6-14 Individual carbon atom is substituted aromatic ring.X is functional group not with azido reaction, and L is suitable leaving group.
In another method for preparing the polymer derivant containing acetylene of the present invention, make that there is about 800Da to arrive About 100,000Da mean molecule quantity, have through protecting functional group or end-capping reagent and in another end on an end The upper PEG polymer with suitable leaving group and acetylene anion contact.
Exemplary reaction process is presented below：
X-PEG-L+-C ≡ CR ' → X-PEG-C ≡ CR ',
Wherein：
PEG is polyethylene glycol and X is the end-capping group such as alkoxy or functional group as described above；And
R ' is H, alkyl, alkoxy, aryl or aryloxy group or substituted alkyl, alkoxy, aryl or aryloxy group.
In example above, when with sufficient concentrations of acetylene anion contact, leaving group L should fully react to undergo The displacement reaction of SN2 types.Those skilled in the art is known to realize that the SN2 of leaving group replaces required reaction by acetylene anion Condition.
Generally in art known method (precipitated product can be included but is not limited to, color is then carried out if necessary Spectrum) realize the purifying of crude product.
Water-soluble polymer can be connected with the FGF-21 polypeptides of the present invention.Water-soluble polymer can be more by being incorporated to FGF-21 Non-naturally encoded amino acids in peptide are non-naturally encoded or any functional group of natural coded amino acid or substituent or addition Connected to any functional group in non-naturally encoded or natural coded amino acid or substituent.Or, water-soluble polymer is logical Cross the naturally occurring amino acid amido of cysteine, lysine or N-terminal residue (including but not limited to) with and have a non-natural The FGF-21 polypeptides connection of coded amino acid.In some cases, FGF-21 polypeptides of the invention comprising 1,2,3,4,5,6,7, 8th, 9,10 alpha-non-natural amino acids, one of them or more than one non-naturally encoded amino acids and water-soluble polymer (including (but It is not limited to) PEG and/or oligosaccharides) connection.In some cases, FGF-21 polypeptides of the invention further comprising 1,2,3,4,5, 6th, 7,8,9, more than the 10 or 10 natural coded amino acids that are connected with water-soluble polymer.In some cases, it is of the invention FGF-21 polypeptides include non-naturally encoded amino acids and one or one that one or more are connected with water-soluble polymer The naturally occurring amino acid being connected more than individual with water-soluble polymer.In certain embodiments, relative to non-junction form, this hair Water-soluble polymer used strengthens the serum half-life of FGF-21 polypeptides in bright.
Number (that is, the Pegylation or sugar for the water-soluble polymer that the adjustable FGF-21 polypeptides with the present invention are connected Base degree) to provide pharmacology, pharmacokinetics or the pharmacodynamic properties of change (including but not limited to increasing or decreasing), example Such as vivo half-life.In certain embodiments, FGF-21 half-life period than unmodified polypeptide increase at least about 10%, 20%th, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 Times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times or at least about 100 times.
PEG derivatives containing strong nucleophilic group (that is, hydrazides, hydrazine, azanol or semicarbazides)
In one embodiment of the invention, with containing the terminal hydrazine being directly connected with PEG main chains, azanol, hydrazides or ammonia The PEG Derivatives Modifieds of base urea part include the FGF-21 polypeptides of the non-naturally encoded amino acids containing carbonyl.
In certain embodiments, PEG derivatives in azanol end will have following structure：
RO-(CH2CH2O)n-O-(CH2)m-O-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1,000 (that is, mean molecule Amount is between 5-40kDa).
In certain embodiments, the PEG derivatives containing hydrazine or containing hydrazides will have following structure：
RO-(CH2CH2O)n-O-(CH2)m-X-NH-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1, and 000 and X is optionally To may be present or non-existent carbonyl (C=O).
In certain embodiments, the PEG derivatives containing semicarbazides will have following structure：
RO-(CH2CH2O)n-O-(CH2)m-NH-C(O)-NH-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1,000.
In another embodiment of the present invention, with containing end azanol, the acyl being connected by means of amido link with PEG main chains The PEG Derivatives Modifieds of hydrazine, hydrazine or semicarbazide moiety include the FGF-21 polypeptides of the amino acid containing carbonyl.
In certain embodiments, azanol end PEG derivatives have following structure：
RO-(CH2CH2O)n-O-(CH2)2-NH-C(O)(CH2)m-O-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1,000 (that is, mean molecule Amount is between 5-40kDa).
In certain embodiments, the PEG derivatives containing hydrazine or containing hydrazides have following structure：
RO-(CH2CH2O)n-O-(CH2)2-NH-C(O)(CH2)m-X-NH-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10, and n is 100-1, and 000 and X is optionally can Present or absent carbonyl (C=O).
In certain embodiments, the PEG derivatives containing semicarbazides have following structure：
RO-(CH2CH2O)n-O-(CH2)2-NH-C(O)(CH2)m-NH-C(O)-NH-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1,000.
In another-individual embodiment of the present invention, spread out with the branch PEG containing terminal hydrazine, azanol, hydrazides or semicarbazide moiety Bio-modification includes the FGF-21 polypeptides of the amino acid containing carbonyl, and wherein branch PEG each chain has in the range of 10-40kDa And can be 5-20kDa MW.
In another embodiment of the present invention, with the PEG Derivatives Modifieds with apparatus derivatorius comprising non-naturally encoded The FGF-21 polypeptides of amino acid.For example, in certain embodiments, hydrazine or hydrazides end PEG derivatives will have following knot Structure：
[RO-(CH2CH2O)n-O-(CH2)2-NH-C(O)]2CH(CH2)m-X-NH-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1,000, and X is optionally To may be present or non-existent carbonyl (C=O).
In certain embodiments, the PEG derivatives containing amino urea groups will have following structure：
[RO-(CH2CH2O)n-O-(CH2)2-C(O)-NH-CH2-CH2]2CH-X-(CH2)m-NH-C(O)-NH-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and X optionally for NH, O, S, C (O) or is not present, and m is 2- 10 and n is 100-1,000.
In certain embodiments, the PEG derivatives containing hydroxyamine groups will have following structure：
[RO-(CH2CH2O)n-O-(CH2)2-C(O)-NH-CH2-CH2]2CH-X-(CH2)m-O-NH2,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and X optionally for NH, O, S, C (O) or is not present, and m is 2- 10 and n is 100-1,000.
The degree and site that water-soluble polymer is connected with FGF-21 polypeptides can adjust FGF-21 polypeptides and FGF-21 polypeptides The combination of acceptor.In certain embodiments, dispose each bonded so that FGF-21 polypeptides (for example exist to combine calibrating by balance Spencer et al., J.Biol.Chem., 263：On described in FGF-21 in 7862-7867 (1988)) measured about 400nM or Below 400nM Kd, with 150nM or below 150nM KdAnd in some cases with 100nM or below 100nM KdWith FGF-21 polypeptide receptors are combined.
The method and chemical descriptor engaged for polymer activation and peptide is in document and in the art To be known.The common method of polymer activation is including but not limited to cyanogen bromide, periodate, glutaraldehyde, bicyclic oxidation Thing (biepoxide), epichlorohydrin (epichlorohydrin), divinylsulfone, carbodiimides, sulfonic acid halide, three chlorotriazines etc. Activating functional group.(referring to R.F.Taylor, (1991), PROTEIN IMMOBILISATION.FUNDAMENTAL AND APPLICATIONS, Marcel Dekker, N.Y.；S.S.Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton；G.T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES, Academic Press, N.Y.；Dunn, R.L. et al. are compiled, POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series, volume 469, American Chemical Society, Washington, D.C.1991).
The functionalization on PEG and some general introductions and the monograph of engagement can be obtained.For example, referring to Harris, Macromol.Chem.Phys.C25：325-373(1985)；Scouten, Methods inEnzymology 135：30-65 (1987)；Wong et al., Enzyme Microb.Technol 14：866-874(1992)；Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9：249-304(1992)；Zalipsky, Bioconjugate Chem.6：150-165(1995).
The method of activated polymer is also seen in WO 94/17039, U.S. Patent No. 5,324,844, WO 94/ 18247th, WO 94/04193, U.S. Patent No. 5,219,564, U.S. Patent No. 5,122,614, WO 90/13540, U.S. In state's patent the 5th, 281,698 and WO 93/15189, and it is following corresponding activated polymer is found in the method that enzyme is engaged In the bibliography of different enzymes, the enzyme includes but is not limited to blood coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrier molecule (U.S. Patent No. 4,412,989), ribalgilase and superoxide dismutase (Veronese Et al., App.Biochem.Biotech.11：141-52(1985)).Cited all bibliography and patent are all to draw Mode is incorporated herein.
Carried out by any facilitated method containing non-naturally encoded amino acids (such as to azido-L-phenylalanine) The Pegylation (that is, adding any water-soluble polymer) of FGF-21 polypeptides.For example, derived with the mPEG blocked through alkynes Thing makes FGF-21 polypeptide Pegylations.Briefly, at room temperature under agitation to containing the FGF-21 to azido-L-Phe Excessive addition solid mPEG (5000)-O-CH in the aqueous solution of polypeptide2-C≡CH.Generally, with the close pH reacted The pK of value (usual pH value is about 4-10)aBuffer solution aqueous buffer solution.For example, suitable in the slow of 7.5 times Pegylations of pH The example of fliud flushing includes but is not limited to HEPES, phosphate, borate, TRIS-HCl, EPPS and TES.If necessary, continuous prison Control and adjust pH value.Reaction is generally set to continue to carry out about 1-48 hours.
Then, reaction product is made to be subjected to hydrophobic interaction chromatogram, so that Pegylation FGF-21 polypeptide variants With free mPEG (5000)-O-CH2- C ≡ CH and when making not block PEG activation at the two ends of molecule so that FGF-21 polypeptides Any high molecular weight component for the Pegylation FGF-21 polypeptides that variant molecules can be formed when being crosslinked is separated.It is hydrophobic Property interaction chromatography during condition should cause free mPEG (5000)-O-CH2- C ≡ CH flow through post, and any crosslinking is poly- PEGylation FGF-21 polypeptide variants compound is then eluted after form needed for realizing and come out, and required form contains one and one The FGF-21 polypeptide variants molecules of individual or more than one PEG group engagement.Suitable condition regards cross-linked composite relative to institute Need the relative size of binding element and change, and be easy to be determined by those skilled in the art.Concentrated by ultrafiltration containing The eluate of required binding element and its desalination is made by filter.
If necessary, one or more kinds of programs it can be further purified as known to those skilled in the art obtained from thin The Pegylation FGF-21 polypeptides of aqueous chromatogram, described program includes but is not limited to affinity chromatography；Anion or cation Exchange chromatography (uses (including but is not limited to) DEAE Ago-Gels)；Silica gel chromatograph；Reversed-phase HPLC；Gel filtration (uses (bag Include but be not limited to) SEPHADEX G-75)；Hydrophobic interaction chromatogram；Size exclusion chromatography；Metal-chelate chromatography；It is super Filter/filter；Ethanol precipitation；Ammonium sulfate precipitation；Chromatofocusing；Displcement chromatography；Electrophoretic procedures (include but is not limited to preparative etc. Electrofocusing)；Differential solubility (including but not limited to ammonium sulfate precipitation)；Or extraction.Can by with globular protein reference material (Preneta, AZ, PROTEIN PURIFICATION METHODS, APRACTICALAPPROACH(Harris and Angal are compiled) IRL Press 1989, 293-306) it is compared by GPC to estimate apparent molecular weight.Tryptose (can be included but is not limited to by proteolytic degradation Enzymatic lysis), the purity of FGF-21-PEG binding elements is analyzed in then mass spectral analysis.Pepinsky RB. et al., J, Pharmcol.& Exp.Ther.297(3)：1059-66(2001).
The water-soluble polymer being connected with the amino acid of the FGF-21 polypeptides of the present invention further can derive without restriction Or be substituted.
PEG derivatives containing azido
In another embodiment of the present invention, it is described with the PEG Derivatives Modified FGF-21 polypeptides containing azide moiety Azide moiety will react with alkynyl moiety present on non-naturally encoded amino acids side chain.In general, PEG derivatives will have In the range of the 1-100kDa and in certain embodiments mean molecule quantity in the range of 10-40kDa.
In certain embodiments, PEG derivatives in azido end will have following structure：
RO-(CH2CH2O)n-O-(CH2)m-N3,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1,000 (that is, mean molecule Amount is between 5-40kDa).
In another embodiment, PEG derivatives in azido end will have following structure：
RO-(CH2CH2O)n-O-(CH2)m-NH-C(O)-(CH2)p-N3,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10, and p is 2-10 and n is 100-1, and 000 (i.e., Mean molecule quantity is between 5-40kDa).
In another embodiment of the present invention, contained with the branch PEG Derivatives Modifieds containing end azide moiety Each chain of the FGF-21 polypeptides of the amino acid of alkynes, wherein branch PEG has in the range of 10-40kDa and can be 5-20kDa's MW.For example, in certain embodiments, PEG derivatives in azido end will have following structure：
[RO-(CH2CH2O)n-O-(CH2)2-NH-C(O)]2CH(CH2)m-X-(CH2)pN3,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10, and p is 2-10, and n is 100-1,000, and And X is optionally O, N, S or carbonyl (C=O), in every case, X may be present or be not present.
PEG derivatives containing alkynes
In another embodiment of the present invention, with the PEG Derivatives Modified FGF-21 polypeptides containing alkynyl moiety, the alkynes Part will react with azide moiety present on non-naturally encoded amino acids side chain.
In certain embodiments, PEG derivatives in alkynes end will have following structure：
RO-(CH2CH2O)n-O-(CH2)m- C ≡ CH,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10 and n is 100-1,000 (that is, mean molecule Amount is between 5-40kDa).
In another embodiment of the present invention, with containing the end nitrine portion being connected by means of amido link with PEG main chains Point or the PEG Derivatives Modifieds of end alkynyl moiety include the FGF-21 polypeptides of the non-naturally encoded amino acids containing alkynes.
In certain embodiments, PEG derivatives in alkynes end will have following structure：
RO-(CH2CH2O)n-O-(CH2)mNH-C(O)-(CH2)p- C ≡ CH,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10, and p is 2-10 and n is 100-1,000.
In another embodiment of the present invention, included with the branch PEG Derivatives Modifieds containing end alkynyl moiety containing folded Each chain of the FGF-21 polypeptides of the amino acid of nitrogen base, wherein branch PEG has in the range of 10-40kDa and can be 5-20kDa MW.For example, in certain embodiments, PEG derivatives in alkynes end will have following structure：
[RO-(CH2CH2O)n-O-(CH2)2-NH-C(O)]2CH(CH2)m-X-(CH2)pC ≡ CH,
Wherein R is simple alkyl (methyl, ethyl, propyl group etc.), and m is 2-10, and p is 2-10, and n is 100-1,000, and And X optionally for O, N, S or carbonyl (C=O) or is not present.
PEG derivatives containing phosphine
In another embodiment of the present invention, with containing activating functional group's (including but not limited to ester, carbonic ester) PEG Derivatives Modified FGF-21 polypeptides, the functional group further comprising will with present on non-naturally encoded amino acids side chain The aryl phosphine groups of azide moiety reaction.In general, PEG derivatives will have in the range of 1-100kDa and in some realities Apply the mean molecule quantity in the range of 10-40kDa in example.
In certain embodiments, PEG derivatives will have following structure：
Wherein n is 1-10；X can be O, N, S or be not present that Ph is phenyl, and W is water-soluble polymer.
In certain embodiments, PEG derivatives will have following structure：
Wherein X can be O, N, S or to be not present, and Ph is phenyl, W be water-soluble polymer and R can be H, alkyl, aryl, Substituted alkyl and substituted aryl.Exemplary R group includes but is not limited to-CH2、-C(CH3)3、-OR′、-NR′R″、- SR ' ,-halogen ,-C (O) R ' ,-CONR ' R " ,-S (O)2R′、-S(O)2NR ' R " ,-CN and-NO2.R ', R ", R " ' and R " " are each solely Hydrogen is on the spot referred to, be substituted or be unsubstituted miscellaneous alkyl, be substituted or is unsubstituted aryl (including but not limited to through 1-3 The aryl of halogen substitution), be substituted or be unsubstituted alkyl, alkoxy or thio alkoxy or aryl alkyl.For example, when this hair When bright compound includes more than one R group, R group is chosen independently of one another, and when having R ', R ", R " ' and R " " During one of group above, these groups are equally chosen independently of one another.When R ' and R " are connected with same nitrogen-atoms, It can combine to form 5 yuan, 6 yuan or 7 yuan of rings with nitrogen-atoms.For example ,-NR ' R " are intended to (but not limited to) 1- pyrrolidines Base and 4- morpholinyls.According to the above-mentioned discussion to substituent, it will be understood by one of ordinary skill in the art that term " alkyl " intends bag Include the group for including the carbon atom combined with the group in addition to hydrogen-based, such as alkylhalide group (including but not limited to-CF3With- CH2CF3) and acyl group (including but not limited to-C (O) CH3、-C(O)CF3、-C(O)CH2OCH3Deng).
Other PEG derivatives and general Pegylation technology
For example, other exemplary the PEG molecules and PEGylation processes that can be connected with FGF-21 polypeptides are including following Molecule and method described in document：U.S. Patent Publication case the 2004/0001838th；No. 2002/0052009；The No. 2003/0162949；No. 2004/0013637；No. 2003/0228274；No. 2003/0220447；2003/th No. 0158333；No. 2003/0143596；No. 2003/0114647；No. 2003/0105275；2003/0105224th Number；No. 2003/0023023；No. 2002/0156047；No. 2002/0099133；No. 2002/0086939；The No. 2002/0082345；No. 2002/0072573；No. 2002/0052430；No. 2002/0040076；2002/th No. 0037949；No. 2002/0002250；No. 2001/0056171；No. 2001/0044526；2001/0021763rd Number；U.S. Patent No. 6,646,110；No. 5,824,778；No. 5,476,653；No. 5,219,564；5,629th, No. 384；No. 5,736,625；No. 4,902,502；No. 5,281,698；No. 5,122,614；No. 5,473,034； No. 5,516,673；No. 5,382,657；No. 6,552,167；No. 6,610,281；No. 6,515,100；6th, No. 461,603；No. 6,436,386；No. 6,214,966；No. 5,990,237；No. 5,900,461；5,739,208th Number；No. 5,672,662；No. 5,446,090；No. 5,808,096；No. 5,612,460；No. 5,324,844；5th, No. 252,714；No. 6,420,339；No. 6,201,072；No. 6,451,346；No. 6,306,821；5,559,213rd Number；No. 5,747,646；No. 5,834,594；No. 5,849,860；No. 5,980,948；No. 6,004,573；6th, 129,912；WO 97/32607、EP 229,108、EP 402,378、WO 92/16555、WO 94/04193、WO94/14758、 WO 94/17039、WO 94/18247、WO 94/28024、WO 95/00162、WO 95/11924、WO 95/13090、WO 95/33490、WO 96/00080、WO 97/18832、WO 98/41562、WO 98/48837、WO 99/32134、WO 99/ 32139、WO 99/32140、WO 96/40791、WO 98/32466、WO 95/06058、EP 439 508、WO 97/03106、 WO 96/21469、WO 95/13312、EP 921 131、WO 98/05363、EP 809 996、WO 96/41813、WO 96/ 07670th, EP 605 963, EP 510 356, EP 400 472, EP 183503 and EP 154 316, it is by reference It is incorporated herein.Any PEG molecules specifically described herein can including but not limited to single-stranded, branched chain, multi-arm chain, list Function, difunctionality, multifunctional form or its any combination of any form are used.
Other polymer and PEG derivatives are described in following patent application case, and the patent application case is all in full The mode of reference is incorporated herein：U.S. Patent Publication case the 2006/0194256th；U.S. Patent Publication case the 2006/th No. 0217532；U.S. Patent Publication case the 2006/0217289th；US provisional patent the 60/755,338th；The U.S. is interim Patent the 60/755,711st；US provisional patent the 60/755,018th；International application PCT/US06/49397 Number；WO 2006/069246；US provisional patent the 60/743,041st；US provisional patent the 60/743,040th；It is international Patent application case the PCT/US06/47822nd；US provisional patent the 60/882,819th；US provisional patent the 60/882nd, No. 500；And US provisional patent the 60/870,594th.
Enhancing is to sero-abluminous compatibility
Also various molecules can be made with the FGF-21 peptide fusions of the present invention to adjust FGF-21 polypeptides partly declining in serum Phase.In certain embodiments, make the FGF-21 polypeptides connection of molecule and the present invention or merge to strengthen to the endogenous blood in animal The compatibility of pure albumen.
For example, in some cases, the restructuring fusion of FGF-21 polypeptides and albumin combination sequence is prepared.Illustrate Property albumin combination sequence include but is not limited to the albumin combination domain (example from streptococcus (streptococcal) Protein G Such as referring to Makrides et al., J.Pharmacol.Exp.Ther.277：534-542(1996)；With Sjolander et al., J, Immunol.Methods 201：115-123 (1997)), or albumin binding peptide, such as in such as Dennis et al., J.Biol.Chem.277：Albumin binding peptide described in 35035-35043 (2002).
In other embodiments, the FGF-21 polypeptides for making the present invention with aliphatic acid are acylated.In some cases, aliphatic acid promotees Enter and sero-abluminous combination.Referring for example to Kurtzhals et al., Biochem.J.312：725-731(1995).
In other embodiments, FGF-21 polypeptides of the invention are directly (to include but is not limited to people with seralbumin Class seralbumin) fusion.Those skilled in the art, which should be understood that, can also make a variety of other molecules and the FGF- of the present invention 21 connection with adjust and seralbumin or other serum components combination.
The glycosylation of X.FGF-21 polypeptides
The present invention includes and has the FGF-21 polypeptides of one or more kinds of non-naturally encoded amino acids with saccharide residue. Saccharide residue can be natural residue (including but not limited to N- acetyl glucosamines) or non-natural residues (including but not limited to 3- Fluorine galactolipin).Sugar can by the glycosidic bond of N or O- connections (including but not limited to N- acetyl galactoses-Serine) or Non-natural key (including but not limited to oxime or the glucosides of corresponding C or S- connections) is connected with non-naturally encoded amino acids.
In vivo or in vitro sugar (including but not limited to glycosyl) can be partly added in FGF-21 polypeptides.At this In some embodiments of invention, with the sugar-modified FGF-21 for including the non-naturally encoded amino acids containing carbonyl as derived from aminooxy group Polypeptide, to produce the corresponding glycosylated polypeptides connected by oxime key., can be by using after sugar is connected with non-naturally encoded amino acids Glycosyl transferase and other ferment treatments further finely to modify sugar to produce the oligosaccharides combined with FGF-21 polypeptides.Referring for example to H.Liu et al., J.Am.Chem.Soc.125：1702-1703(2003).
In some embodiments of the invention, directly there is the poly- of regulation structure to prepared by aminooxy group derivative form The sugar-modified FGF-21 polypeptides for including the non-naturally encoded amino acids containing carbonyl.Those skilled in the art should be understood that can Sugar is set to be connected with non-naturally encoded amino acids using other functional groups (including azide, alkynes, hydrazides, hydrazine and semicarbazides).
In some embodiments of the invention, (including but is not limited to) Hughes's root [3+2] cycloaddition reaction can then be passed through Respectively the non-naturally encoded amino containing azido or alkynyl is included with (including but is not limited to) alkynyl or azido derivative modification The FGF-21 polypeptides of acid.This method allows with high selective modification protein.
XI.FGF-21 dimers and polymer
The present invention also provides the combination of FGF-21 and FGF-21 analogs, and for example homodimers, heterodimer, homotype are more Polymers or heteromultimer thing (that is, trimer, tetramer etc.), wherein containing one or more non-naturally encoded amino acids FGF-21 is directly combined with polypeptide backbone, or by connexon and another FGF-21 or its FGF-21 variant or non-FGF-21 or Any other polypeptide of its FGF-21 variant is combined.Due to single phase than molecular weight increase, so FGF-21 dimers or Polymer binding element can show novel or required property, including but not limited to show different pharmacology relative to monomer FGF-21 , pharmacokinetics, pharmacodynamic properties, adjusted treatment half-life period or adjusted plasma half-life.In some embodiments In, FGF-21 dimers of the invention will adjust the signal transduction of FGF-21 acceptors.In other embodiments, FGF- of the invention 21 dimers or polymer will serve as the antagonist, activator or conditioning agent of FGF-21 acceptors.
In certain embodiments, one or more FGF- present in the dimer or polymer containing FGF-21 21 molecules include the non-naturally encoded amino acids being connected with water-soluble polymer.
In certain embodiments, FGF-21 polypeptides are (including but is not limited to) to pass through Asn-Lys amido links or Cys-Cys bis- Sulfide linkage is directly connected to.In certain embodiments, FGF-21 polypeptides and/or the non-FGF-21 molecules of connection will include different non-days Right coded amino acid is to promote dimerization, and it includes but is not limited to a non-naturally encoded amino acids of the first FGF-21 polypeptides In alkynes will be connect with the azide in dimolecular second non-naturally encoded amino acids by Hughes's root [3+2] cycloaddition Close.Or, the non-FGF-21 molecules of FGF-21 and/or connection comprising the non-naturally encoded amino acids containing ketone can with comprising containing hydroxyl The second polypeptide engagement of the non-naturally encoded amino acids of amine, and the polypeptide is reacted by forming corresponding oxime.
Or, the non-FGF-21 molecules of two kinds of FGF-21 polypeptides and/or connection are connected by connexon.It can be used any Special-shaped bifunctional linker or homotype bifunctional linker connect the non-FGF-21 molecules of two kinds of molecules and/or connection, and it can With identical or different primary sequence.In some cases, for FGF-21 and/or connection non-FGF-21 molecules to be connected Connexon together can be bifunctional PEG reagent.Connexon can have various molecular weight or molecular length.Can be used it is larger or The connexon of relatively small molecular weight FGF-21 and connection entity between FGF-21 and its acceptor or connection entity with It provides required spatial relationship or conformation between combining collocation thing (if present).It can also be used with longer or shorter point The connexon of sub- length is between FGF-21 and connection entity or in connection entity collocation thing (if present) in connection Between provide needed for space or pliability.
In certain embodiments, the present invention provides the water-soluble bifunctional linker with dumbbell structure, the structure bag Include：A) azido, alkynes, hydrazine, hydrazides, azanol or the part containing carbonyl at least one first end of main polymer chain； And b) at least one second functional group in the second end of main polymer chain.Second functional group can be with first functional group's phase It is same or different.In certain embodiments, second functional group not with the first functional group reactionses.In certain embodiments, the present invention is carried For the water soluble compound of at least one arm comprising branched molecule structure.For example, branched molecule structure can be dendroid.
In certain embodiments, the present invention provides through with formed by the reaction of water-soluble activated polymer comprising one or The polymer of more than one FGF-21 polypeptide, the water-soluble activated polymer has following structure：
R-(CH2CH2O)n-O-(CH2)m- X,
It about 5 to 3,000, m is 2-10 that wherein n, which is, X can for azido, alkynes, hydrazine, hydrazides, aminooxy group, azanol, acetyl group or Part containing carbonyl, and R for can be identical or different with X end-capping group, functional group or leaving group.R can be selected from by The functional group of the group of consisting of：Hydroxyl, through protect hydroxyl, alkoxy, N-hydroxy-succinamide ester, 1- BTAs Ester, carbonic acid N-hydroxy-succinamide ester, carbonic acid 1- BTAs ester, acetal, aldehyde, aldehydrol, alkenyl, acrylate, first Base acrylate, acrylamide, active sulfone, amine, aminooxy group, through protecting amine, hydrazides, through protecting hydrazides, through protecting mercaptan, carboxylic Acid, through protect carboxylic acid, isocyanates, isothiocyanates, maleimide, vinyl sulfone, dithiopyridines, vinylpyridine, Iodoacetamide, epoxides, glyoxal, diketone, methanesulfonates, tosylate and trifluoro esilate, alkene and ketone.
The measurement of XII.FGF-21 polypeptide actives and FGF-21 polypeptides to the compatibility of FGF-21 polypeptide receptors
Show that FGF-21 stimulates the glucose uptake in 3T3-L1 fat cells and strengthens insulin sensitivity, it is described 3T3-L1 fat cells are such as U.S. Patent Publication case the 20040259780th (it is incorporated herein in entirety by reference) Example 3 shown in be used for study adipose tissue metabolism vitro model.Diabetes B is characterised by Various Tissues type Glucose uptake in (including adipose tissue) is not enough.Therefore, FGF-21 is applied to treat 2 type glycosurias because of reduction blood sugar level Disease.In addition, FGF-21 because by faster and more effectively glucose utilization come increase energy expenditure and be applied to treatment obesity Disease.In addition, shown that FGF-21 stimulates the glucose uptake in 3T3-L1 fat cells in insulin-dependent mode, this expression It is also applied for treating type 1 diabetes.Referring to U.S. Patent Publication case the 20040259780th.U.S. Patent Publication case Show FGF-21 under the insulin (5nM) of suboptimum concentration and in the feelings in the absence of insulin in No. 20040259780 The glucose uptake in 3T3-L1 fat cells is stimulated with concentration dependant manner under condition.In addition, FGF-21 is public in United States Patent (USP) Induced glucose in the in vitro tissue model described in case the 20040259780th is opened to absorb.
The glucose uptake in following methods analysis 3T3-L1 fat cells can be used.3T3-L1 cells are to be obtained from U.S.'s allusion quotation Type DSMZ (ATCC, Rockville, Md.).Her grignard culture medium (Dulbecco ' is improved in Du's shellfish Cattell Smodified Eagle ' s medium) in contain culture in the growth medium (GM) of the hyclone rich in 10% iron thin Born of the same parents.On standard Adipocyte Differentiation, reached in cell after covering with and (being referred to as the 0th day) two days, be exposed to cell and contain 10% hyclone, 10 μ g/ml insulin, 1 μM of dexamethasone (dexamethasone) and 0.5 μM of isobutyl methylxanthine Differential medium (DM) in up to 48 hours.Then, cell is maintained at containing 10% hyclone and 10 μ g/ml insulin Break up in wild Oryza species.The in vitro efficiency of glucose uptake calibrating measurement known to those skilled in the art can be used.Live body Outer efficiency may be defined as measuring for glucose uptake of the FGF-21 compounds in the calibrating based on cell, and be FGF chemical combination The biopotency of thing is measured.It is represented by EC50, EC50To produce the compound of 50% activity in single dose reaction experiment Valid density.
Glucose transport examines and determine (insulin-dependent)：Such as by 0.1mM 2- deoxidations-D- [14C] accumulation of glucose examined It is fixed, following measurement hexose intake：With being warming up to 37 DEG C and KRP buffer solutions (136mM NaCl, 4.7mM containing 0.2%BSA KCl、10mM NaPO4、0.9mM CaCl2、0.9mM MgSO4, pH 7.4) and the 3T3-L1 fat cells in 12 orifice plates are washed two It is secondary, cultivated 2 hours in the Leibovitz ' s L-15 culture mediums containing 0.2%BSA in air indoors at 37 DEG C, with containing The KRP for having 0.2%BSA buffer solutions is washed twice again, and at 37 DEG C indoors in air in the absence of (only Me2SO) or Cultivated 30 minutes in KRP, 0.2%BSA buffer solution in the case of there is wortmannin (wortmannin).Then, by pancreas Island element increased to 100nM ultimate density up to 15 minutes, and last 4 minutes measurement 2- deoxidations-D- [14C] glucose takes the photograph Take.The non-specific uptake measured in the presence of 10 μM of cytochalasin Bs (cytochalasin B) is subtracted from all values.With Pierce bicinchoninic acids (bicinchoninic acid) calibrating determines protein concentration.For each experiment, a usual formula three Part or quadruplicate measurement intake.Can study in the presence of insulin 3T3-L1 fat cells are carried out with FGF-21 it is acute and slow Property pretreatment effect.
Glucose transport examines and determine (insulin-dependent)：3T3-L1 fibroblasts are applied in 96 orifice plates and are divided into Fat cell was up to 2 weeks.After differentiation, it is made to suffer from hunger and be handled 24 hours with FGF-21 in the culture medium without serum. After processing, cell is washed twice with the KRBH buffer solutions containing 0.1%BSA immediately.In labeled glucose (no pancreas islet Element) in the presence of glucose uptake is carried out in KPBH buffer solutions.Show FGF-21 under the insulin (5nM) of suboptimum concentration And stimulate the glucose uptake in 3T3-L1 fat cells (to join with concentration dependant manner in the case of in the absence of insulin See U.S. Patent Publication case the 2004259780th).In addition, having shown the FGF-21 polypeptid induction in vitro tissue models of the present invention In glucose uptake.
In in vitro glucose transport model, glucose transport calibrating is described as follows：Krebs-Henseleit buffer solutions are stored up Standby solution-storing solution 1：NaCl(1.16M)；KCl(0.046M)；KH2PO4(0.0116M)；NaHCO3(0.0253M).Storing solution 2：CaCl2(0.025M)；MgSO4(2H2O)(0.0116M).BSA：Using ICN Cohn part V, i.e., completely without dialysis not The BSA of fatty acids.It is prepared by culture medium：To 395ml dH250ml Krebs storing solutions 1 are added in O and 95%O is used2/ 5% CO2Ventilation 1 hour.Add 50ml storing solutions 2 and use dH2O supplies 500ml.Add the BSA of 500mg ICN not fatty acids. Pre-incubation and cultivation culture medium：32mM mannitols, 8mM glucose.Wash culture medium：40mM mannitols, 2mM pyruvic acid Salt.Transport culture medium：39mM mannitols, 1mM 2-DG；32mM mannitols, 8mM 3-O-MG.Insulin solutions：(pig pancreas Island element [Lilly] 100,000,000 μ U/ml), ultimate density is 2000 μ U/ml or 13.3nM.Radioactive label culture medium system It is standby：Specific activity used：2DG=1.5mCi/ml；3-O-MG=37 μ Ci/ml；Or the μ Ci/m of mannitol=8.Used per 100g body weight 0.1cc amobarbitals (Nembutal) anesthetized rat.Cut off musculature and use 0.9% normal saline flushing, then 29 It is placed at DEG C in pre-incubation culture medium (2ml) up to 1 hour.Musculature is transferred to cultivation culture medium (2ml；Except including pancreas It is identical with pre-incubation culture medium outside island element or test compound) in and cultivate 30 minutes (depend on experiment condition).Then, Musculature was transferred in washing culture medium (2ml) up to 10 minutes at 29 DEG C, mark culture medium (1.5ml) is subsequently transferred to In up to 10 minutes (3-O-MG) or 20 minutes (2DG).Musculature is cut, weighs and is placed into the PA tube on dry ice. 1ml1N KOH are added in pipe, then pipe is placed into 70 DEG C of water-baths and reached 10-15 minutes, pipe is vortexed every several minutes. Managed described in cooled on ice and add 1ml1N HCl, be then sufficiently mixed.Then, 200 μ l supernatants are placed into dual multiple sudden strain of a muscle In bright bottle and counted (compared with known radioactive standard thing) with scintillation counter.
On shrinking, muscle is cultivated 1 hour in pre-incubation/cultivation culture medium first.After 1 hour, by each to (every Rat a pair) in one piece of muscle follow closely new cultivation culture flash be transferred on stimulating apparatus and by another piece of muscle In.The muscle shunk is stimulated with 70Hz 200 milliseconds of wave trains, each pulse wherein in the wave train is 0.1 millisecond.Under 10-15V with Once per second transmitted the wave train up to 2 × 10 minutes, had stop therebetween within 1 minute.At the end of stimulation period, flesh is removed from stimulating apparatus Meat and be placed into washing culture medium in up to 10 minutes, be then placed into mark culture medium as outlined above.
Technology known to those skilled in the art and method can be used to prepare FGF receptor.Standard or can be used That knows in vitro or in vivo examines and determine to determine FGF-21 polypeptide actives.For the non-Pegylation comprising alpha-non-natural amino acid Or for Pegylation FGF-21 polypeptides, can be by using BIAcoreTMBiology sensor (Pharmacia) measures FGF- The compatibility of 21 pairs of its acceptors.
No matter which kind of method to produce FGF-21 polypeptides using, the FGF-21 polypeptides is subjected to bioassay. In general, biological activity test should provide the analysis to required result, the increasing or decreasing of such as bioactivity (with through modification FGF-21 compare), different bioactivity (compared with the FGF-21 through modification), acceptor or the compatibility point for combining collocation thing Analysis, FGF-21 in itself or its acceptor conformation or structure change (compared with the FGF-21 through modification) or serum half-life analysis.
Above-mentioned compilation for the bibliography of calibration method is simultaneously not exhaustive, and those skilled in the art will recognize The calibrating of final result needed for other being applied to test.
XIII. the measurement of efficiency, feature vivo half-life and pharmacokinetic parameter
The importance of the present invention is by being engaged in polypeptide with water-soluble polymer portion or asynthetic situation Lower structure FGF-21 polypeptides and the biological half-life of extension obtained.It is quick after the administration of FGF-21 polypeptide serum-concentrations to reduce So that assessing to not engaging with engagement and the biological respinse that FGF-21 polypeptides and its variant treated and becoming important.For example After subcutaneous or Intravenous administration, engagement of the invention can also have the blood of extension with FGF-21 polypeptides and its variant is not engaged Clear half-life period, so that measuring and being possibly realized for example, by ELISA method or preliminary screening calibrating.It can be used from business ELISA the or RIA kits in source.The measurement of in vivo biological half-life is carried out as described herein.
Scheme the FGF-21 comprising non-naturally encoded amino acids can be determined according to known to those skilled in the art The efficiency and feature vivo half-life of polypeptide.
It can be assessed in normal Sprague-Dawley male rats (each N=5 animal for the treatment of group) and include non-natural The pharmacokinetic parameter of the FGF-21 polypeptides of coded amino acid.Animal is by the single dose through every μ g of rat 25 of intravenous receiving Or through subcutaneously receiving every μ g of rat 50 single dose, and about 5-7 blood sample will be gathered according to predetermined time-histories, for not with water For the FGF-21 polypeptides comprising non-naturally encoded amino acids of soluble polymer engagement, about 6 hours are generally lasted；And for For the FGF-21 polypeptides engaged comprising non-naturally encoded amino acids and with water-soluble polymer, about 4 days are generally lasted.Can be direct By the pharmacokinetic data of the FGF-21 without non-naturally encoded amino acids and on including non-naturally encoded amino acids The data that FGF-21 polypeptides are obtained are compared.
Also pharmacokinetic parameter can be assessed in primate (for example, machin).Generally, through subcutaneously or through vein Interior administration single injection liquid, and with time monitoring serum FGF-21 levels.
The specific activity of the FGF-21 polypeptides of the present invention can be determined by known various calibratings in art.It can pass through Method known to described herein or reference or those skilled in the art is obtained and purified according to the present invention to test The bioactivity of FGF-21 polypeptide muteins or its fragment.
The polypeptide of the present invention can be used to suffer from adult-onset diabetes (NIDDM to treat：2 types), insulin according to Rely the mammal of property diabetes (1 type) and obesity, glucose clearance deficiency, hyperglycemia, hyperinsulinemia etc..Such as Shown in U.S. Patent Publication case the 20040259780th (it is incorporated herein in entirety by reference), FGF-21 is in sugar In the animal model of urine disease and obesity effectively.Because the metabolic profile of the various animal models of obesity and diabetes is present not Together, so being analyzed multiple models to distinguish hyperinsulinemia, hyperglycemia and the effect of obesity.Diabetes And fat (ob/ob) mouse is characterised by huge fertile disease (massive obesity), binge eating (db/db) (hyperphagia), changeability hyperglycemia, insulin resistance, hyperinsulinemia and heat production it is impaired (Coleman, Diabetes 31：1,1982；E.Shafrir, Diabetes Mellitus；H.Rifkin and D.Porte, Jr. are compiled, (ElsevierScience Publishing Co., Inc., New York, the 4th edition, 1990), the 299-340 pages).However, Even more serious (Coleman, the Diabetes 31 of diabetes in db/db models：1,1982；E.Shafrir, Diabetes Mellitus；H.Rifkin and D.Porte, Jr. are compiled, (Elsevier Science Publishing Co., Inc., New York, the 4th edition, 1990), the 299-340 pages).Zucker (fa/fa) rats severe obesity, with hyperinsulinemia and With insulin resistance (Coleman, Diabetes 31：1,1982；E.Shafrir, Diabetes Mellitus； H.Rifkin and D.Porte, Jr. are compiled, (Elsevier Science Publishing Co., Inc., New York, the 4th edition, 1990), the 299-340 pages), and fa/fa mutation can for muroid db be mutated rat it is equivalent mutation (Friedman et al., Cell 69：217-220,1992；Truett et al., Proc.Natl.Acad.Sci.USA 88：7806,1991).Tubby (tub/tub) feature of mouse is obesity, medium insulin resistance and hyperinsulinemia, and without notable hyperglycemia (Coleman et al., J.Heredity 81：424,1990).
Also monosodium glutamate (MSG) model (Olney, the Science164 of the obesity chemically induced can be studied： 719,1969；Cameron et al., Cli.Exp.Pharmacol.Physiol.5：41,1978), wherein obesity is not as heredity It is serious and do not produce binge eating, hyperinsulinemia and insulin resistance in model.Finally, it can test and chemically induce Diabetes Streptozotocin (streptozotocin；STZ) model is to study the high blood in the case of in the absence of obesity The effect of sugared disease.The animal treated with STZ lacks insulin and severe hyperglycemia (Coleman, Diabetes 31：1, 1982；E.Shafrir, iabetes Mellitus；H.Rifkin and D.Porte, Jr. are compiled, (ElsevierScience Publishing Co., Inc., New York, the 4th edition, 1990), the 299-340 pages).
The FGF-21 polypeptides of the present invention can be in vivo assessed in Septic Shock Model in ob/ob mouse.Referring to U.S. State's patent publication the 20050176631st, it is to be incorporated herein in entirety by reference.
XIV. offer medicine and medical composition
The polypeptide or protein of the present invention is (including but not limited to comprising one or more alpha-non-natural amino acids FGF-21, synzyme, protein etc.) optionally (include but is not limited to) combine for therapeutical uses with suitable pharmaceutical carrier. For example, compound of these compositions comprising therapeutically effective amount and pharmaceutically acceptable supporting agent or excipient.The supporting agent Or excipient includes but is not limited to physiological saline, buffer saline, dextrose, water, glycerine, ethanol and/or its combination.System The standby composite for being applied to dispensing pattern.In general, administration method of protein known to those skilled in the art, and Methods described can be used for the polypeptide of the administration present invention.
The method according to known to those skilled in the art, optionally in one or more kinds of appropriate work of disease It is external and/or in vivo in animal model polypeptides of the test bag containing one or more kinds of present invention therapeutic combination so that Confirm effect, tissue metabolism and estimate dosage.Specifically, alpha-non-natural amino acid homologue phase herein can initially be passed through (including but not limited to include the FGF- of one or more alpha-non-natural amino acids through modification for natural amino acid homologue The comparison of 21 polypeptides and natural amino acid FGF-21 polypeptides) activity, stability or other suitably measure (that is, examined related In fixed) determine dosage.
Offerd medicine by being generally used for guiding molecule with any approach that blood or histocyte are finally contacted.The present invention Non-natural amino acid polypeptides be optionally together with one or more kinds of pharmaceutically acceptable supporting agents with any suitable Mode administration.The appropriate method of the polypeptide into patient's administration the context of the invention can be used, although and can be used Carry out administration particular composition more than a kind of approach, but particular approach can generally be provided more instant than another approach and effectively acted on Or reaction.
Pharmaceutically acceptable supporting agent is part by the particular composition of desire administration and the spy for administration composition Determine method decision.Accordingly, there exist a variety of suitable composites of the medical composition of the present invention.
The FGF-21 polypeptides of the present invention can be by any conventional route suitable for protein or peptide come administration, the approach It is including but not limited to parenteral, for example (include but is not limited to) subcutaneous or intravenous injection or the injection or defeated of any other form Liquid.Oral, intravenous, intraperitoneal, muscle can be included but is not limited to by number of ways administration peptide composition, the approach It is interior, percutaneous, subcutaneous, local, sublingual or rectal.Include the combination through modification or unmodified non-natural amino acid polypeptides Thing can also pass through liposome administration.The commonly known dosing way of those skilled in the art and appropriate composite. FGF-21 polypeptides can be used alone or are applied in combination with other suitable components (such as pharmaceutical carrier).FGF-21 polypeptides can be with it He is applied in combination medicament (including but not limited to oral antidiabetic).
Term " antidiabetic " should represent to can be used for treating, preventing any glucose metabolism disorder or its any complication (including any symptom, disease or complication specifically described herein) or otherwise reduce any medicine of its seriousness.It is anti- Rezulin includes insulin, thiazolidinedione, sulfonylureas, benzoic acid derivative, Alpha-glucosidase inhibitor etc..It can be this hair The antidiabetic of other general classes of a part for bright composition includes (term defined in quotation marks)：Official US's pharmacopeia Or " medicine " approved in Official US's NF (or its any annex)；What U.S. FDA was ratified is " new drug " and " new Veterinary drug ", these terms are used in United States Code No. 21；(" criticized in the U.S. or the overseas any medicine for needing government entity to ratify Quasi- medicine ")；Regulatory approved must be obtained with any medicine (" regulatory approved medicine ") in accordance with 21U.S.C. § 355 (a)；Used as people Medicine application (according to 21U.S.C. § 379 (g)) or any medicament for just entering pedestrian's medication application (according to 21U.S.C. § 379 (g)) (" people's medication ").(the original application date referred to all referring to present application of all legal code names on this definition Code name.) other antidiabetics disclose in this article, and known to those skilled in the art.It is many in art The well known current medical or antidiabetic for being used to control diabetes B includes multiple species：Biguanides, thiazolidinedione, Sulfonylurea, benzoic acid derivative and alpha-glucosidase inhibitors.These medicines generally have different binding modes.Think biguanides Class (such as melbine (metformin)) can prevent excessive hepatic gluconeogenesis.Think thiazolidinedione by increasing periphery Glucose processing speed and work.Sulfonylurea is (for example, orinase (tolbutamide) and glibenclamide ) and benzoic acid derivative (such as Repaglinide (repaglinide)) is dropped by stimulating insulin secretion (glyburide) Low plasma glucose.Alpha-glucosidase inhibitor Reverse transcriptase makes the alpha-Glucosidase of carbohydrate metabolism, so as to postpone Carbohydrate absorption simultaneously weakens hyperglycaemia after meals.It additionally, there are a variety for the treatment of sugar for not yet getting the Green Light and being used for the mankind Urinate the suggestion therapy of disease.
It is known that for controlling the current of diabetes and its predecessor's syndrome (such as insulin resistance) in art Medicine or antidiabetic include five class compounds：Biguanides, such as melbine；Thiazolidinedione；Sulfonylurea, for example, Orinase and glibenclamide；Benzoic acid derivative, such as Repaglinide；And alpha-glucosidase inhibitors.In addition to these medicaments, A variety of other therapeutic agents also can be with FGF-21 polypeptides in combination of the invention using to improve glucose control, and the therapeutic agent includes (but not limited to) DPP-4 inhibitor.Some of these antidiabetics medicine has got the Green Light and used for the mankind.Clinical test The leading DPP-4 compounds of middle test include vildagliptin (Vildagliptin) (higher-dimension this (Galvus)) (LAF237), west Ta Lieting (Sitagliptin) (Jia Luweiya (Januvia)), BMS-477118 (Saxagliptin) and Egelieting (Alogliptin).The U.S. is used to treat diabetes B in approval Jia Luweiya on October 17th, 2006 (sitagliptin), and it is used Make single current system method or the combination treatment combined with melbine or thiazolidinedione.Through 4 week period diabetes B was suffered to 93 Patient (average HbAlc is 7.4%) administration first generation Novartis (Novartis) compound 1- [[[2- [(5- cyanopyridine -2- bases) Amino] ethyl] amino] acetyl group] -2- cyano group-(S)-pyrrolidines (NVP DPP728), so as to reduce blood in 4 weeks study periods Starch glucose, insulin and HbAlc level.Referring to Inhibition of Dipeptidyl Peptidase IV Improves Metabolic Control Over a 4-Week StudyPeriod in Type 2 Diabetes.Diabetes Care.2002 May；25(5)：869-875.It is also noted that the patient for receiving melbine is building Show additional glucose reduction benefit after vertical GLP-1 therapies.Referring to Additiveglucose-lowering effects of The diabetes.Diabetes Care.2001 of glucagon-like peptide-1 and metformin in type 2 April；24(4)：720-5.In the research of 10 fat non-diabetic male patients, the administration of melbine and oral grape Sugar load Posterior circle GLP-1 level increase is relevant, and in the experiment using pooled human plasma, in existence or non-existence In the case of DPP-4, after being cultivated 30 minutes at 37 DEG C, melbine (0.1-0.5 mcg/mls) significantly inhibits GLP-1 (7- 36) degraded of acid amides.The author of the research proposes that melbine can be in vitro with vivo suppressing GLP-1 enzymatic decomposition Possibility.Referring to (GLP-1) the and leptin of Effect of metformin onglucagon-like peptide 1 Levels in obese nondiabetic subjects.DiabetesCare.2001 March；24(4)：489-94.Use In vitro biochemical analysis carries out the analysis of the relation between DPP-4 and GLP-1 degradeds.Demuth and colleague use multiple next The mankind DPP-4 in source has found that the GLP-1 degradeds that melbine is mediated on DPP-4 have no influence.Referring to Metformin Effects on Dipeptidylpeptidase IV Degradationof Glucagon-like Peptide-1.Biochem Biophys Res Commun.2002 March 15；291(5)：1302-8.
In the biguanides of Remedies for diabetes is suitable for, has proven to melbine and especially succeed.Melbine (N, N- bis- The carbodiimide diamides (N, N-DIMETHYLIMIDODICARBONIMIDICDIAMIDE) of methyl imide two；1,1- dimethyl Biguanides；N, N- dimethylbiguanide；N, N- dimethylbiguanide；N '-dimethylguanine base guanidine) it is by reducing the glucose of liver The antidiabetic for producing and reducing the intestinal absorption of glucose and work.It is also considered as its group for improveing other positions in body The insulin sensitivity (increase peripheral glucose uptake and utilization) knitted.Melbine improvement Abnormal glucose tolerance (IGT) is individual With the plasma glucose after meals before the glucose-tolerant of body and diabetes B individual, reduction meals.In the absence of insulin In the case of, melbine is generally invalid.Bailey, Diabetes Care 15：755-72(1992).
If effect of melbine is shown in dry test.In one carried out to medium fat diabetes B patient In item research, after the Metformin therapy that is independent or being combined with sulfonylureas of 29 weeks, HbAlc levels are improved to from 8.6% 7.1%.DeFronzo et al., New Engl.J.Med.333：541-49(1995).Melbine also has to serum lipids Profit influence, so as to reduce serum triglyceride average value on an empty stomach, T-CHOL and LDL-C level and show to it Its lipid level has no adverse effect.In another experiment, melbine improves the blood glucose of NIDDM individuals with dose-responsive manner Control.After 14 weeks, daily 500mg and 2000mg melbine makes HbAlc reduce by 0.9% and 2.0% respectively.Garber et al., Am J.Med.102：491-97(1997).Melbine also can have favourable control to the non-diabetic patients of insulin resistance Treatment is acted on.One research shows, can be reduced blood pressure with the fat non-diabetic women of Or Metformin In Treating hypertension, empty stomach and Portugal The plasma insulin fibrinogen that grape sugar is stimulated.Giugliano et al., Diabetes Care 16：1387-90(1993).
Generally with Metformin hydrochloride form administration melbine.Covering makes this form and all other service form Melbine with the present invention FGF-21 polypeptides be used together.Generally, with Metformin hydrochloride or any other pharmacological reagent The fixed dosing regimen of hyperglycaemia in processing diabetic should individually take in.Dosage should be according to validity and tolerance Property individually takes in, but typically not greater than 2550mg maximum recommended daily dose.
The thiazolidinedione being intended for use in during the present invention is implemented includes troglitazone (troglitazone) etc..It is well-known Such compound, for example, as U.S. Patent No. 5,223,522, the 5th, 132, No. 317, the 5th, 120, No. 754, the 5th, 061, No. 717, No. 4,897,405, No. 4,873,255, No. 4,687,777, No. 4,572,912, No. 4,287,200 and No. 5,002,953 and Current Pharmaceutical Design 2：Described in 85-101 (1996).Troglitazone It is a kind of receptor-gamma (PPAR γ) that may be activated by activation of Peroxisome proliferation and increases glucose transport Oral antihyperglycemic agent thing.By such activation, troglitazone can strengthen the expression of GLUT4 glucose transporters, so that The glucose uptake increase of insulin stimulating.Troglitazone may also weaken the activation of gluconeogenesis and/or sugar solution.
HbAlc is the blood testing for measuring glycosylated amount, and when patient experience blood glucose increased period, HbAlc is usual It is higher.The test provides the assessment to the blood glucose control of last 2-3 months of patient.The blood produced by troglitazone therapy Sugar control makes HbAlc reduce about 1% to 2%.Mimura et al., Diabetes Med.11：685-91(1994)；Kumar etc. People, Diabetologia 39：701-09(1996).May not appearance effect after therapy several weeks are started.Troglitazone also may be used Insulin requirements can be reduced.In to an experiment with NIDDM and using patient's progress of exogenous insulin, for dosage For troglitazone for 200mg and 600mg, average HbAlc reduces by 0.8% and 1.4% respectively.Insulin requirements reduction is up to 29%.Schwartz et al., New Engl.J.Med.338：861-66(1998).To being arranged using the bent lattice of 400mg and 600mg In another research that the NIDDM diabetics of ketone are carried out, glucose level is all reduced after fasting glucose level and meals, And hyperinsulinism-positive glucose clamp (hyperinsulinemiceuglycemic clam) shows that glucose processing is higher than Pretreatment level about 45%.Maggs et al., Ann.Intern.Med.128：176-85(1998).
In one is studied, the Portugal of obese patient of the 400mg troglitazones increase with exception or normal glucose tolerance Grape sugar processing speed.Nolan et al., New Eng.J.Med 331：1188-93(1994).To with IGT and with gestation In another research that the women of phase diabetic history is carried out, the improvement insulin dynamic equilibrium of 600mg troglitazones, including improvement pancreas Island element sensitiveness simultaneously reduces circulation insulin concentration, but glucose-tolerant does not change.Berkowitz et al., Diabetes 45： 172-79(1996).Thiazolidinedione is available for NIDDM risks colony (such as the women with POCS or GDM) to use, with pre- Anti- or delay NIDDM breaking-out.U.S. Patent No. 5,874,454.Effective dose when troglitazone is used alone is in daily agent In the range of amount is about 10mg to about 800mg, and can suitable scope be intended to be used in the present invention.Except with melbine Outside being used together, troglitazone can also be applied in combination with insulin and sulfonylureas agent.Referring for example to U.S. Patent No. 5,859, No. 037.
Sulfonylureas is generally worked by increasing the release of insulin in pancreas so as to reduce plasma glucose.It is specific next Say, sulfonylureas is worked by blocking ATP sensitive potassium channels.Sulfonylureas Glimepiride (glimepiride) can also pass through Stimulate the migration of GLUT4 transporters and increase insulin sensitivity.When HbAlc is higher than 8%, generally prescribed with sulfonylureas. Referring also to U.S. Patent No. No. 5,258,185, No. 4,873,080.
Sulfonylureas is well-known class compound in art, for example, such as U.S. Patent No. 3,454,635, No. 3,669,966, No. 2,968,158, No. 3,501,495, No. 3,708,486, No. 3,668,215, the 3rd, Described in No. 654,357 and No. 3,097,242.It is intended for use in the exemplary sulfonylureas (circle in certain embodiments of the present invention Typical daily dose is indicated in bracket) include Acetohexamide (acetohexamide) (in about 250mg to about 1500mg scopes It is interior), chlorpropamide (chlorpropamide) (in the range of about 100mg to about 500mg), tolazamide (tolazimide) (in the range of about 100mg to about 1000mg), orinase (in the range of about 500mg to about 3000mg), gliclazide (gliclazide) (in the range of about 80mg to about 320mg), Glipizide (glipizide) is (in about 5mg to about 40mg scopes It is interior), Glipizide GITS (in the range of about 5mg to about 20mg), glibenclamide (in the range of about 1mg to about 20mg), micron meter Very littleization glibenclamide (in the range of about 0.75mg to about 12mg), Glimepiride (in the range of about 1mg to about 8mg), AG-EE 623ZW etc..Glimepiride is first antidiabetic being used together with insulin that gets the Green Light in such compound, and Reduced to it using related risk of hypoglycemia.
A variety of Alpha-glucosidase inhibitors can be used in conjunction with the invention to treat and/or prevent diabetes.It is described to suppress Agent Reverse transcriptase makes the alpha-Glucosidase of carbohydrate metabolism, so as to postpone carbohydrate absorption and weaken high after meals Blood glucose.Clissod et al., Drugs 35：214-23(1988).It can show by reducing HbAlc levels to reduce glucose.Beat The exemplary Alpha-glucosidase inhibitor calculated in implementing for the present invention includes acarbose (acarbose), Miglitol (miglitol) etc..The effective dose of acarbose and Miglitol is all in the range of about 25mg to about 300mg daily.
Alpha-glucosidase inhibitor can be used together with the polypeptide and sulfonylureas of the present invention.Show that alpha-Glucosidase presses down Preparation is combined with independent sulfonylureas makes HbAlc levels typically be reduced to 1.0% from about 0.5%.In addition, having shown alpha-Glucosidase Inhibitor can effectively reduce post-meal blood glucose rise.Lefevre et al., Drugs 44：29-38(1992).
A variety of benzoic acid derivatives can be used together to treat and/or prevent diabetes with the polypeptide of the present invention.These medicines Agent (also referred to as meglitinide (meglitinide)) is the non-sulfonylureas hypoglycemia agent with insulin secreting ability.Citing comes Say, Repaglinide seems to be combined with the ATP sensitive potassium channels on pancreatic beta cell and so as to increase insulin secretion.Intend Exemplary benzoic acid derivative in implementing for the present invention is including Repaglinide etc..On Repaglinide, effective daily dose can In the range of about 0.5mg to about 16mg, and it can take medicine before the meal every time.
Currently study a variety of medicaments as potential mankind's antidiabetic.When available for therapeutical uses, appoint What such medicament can all be used together to treat and/or prevent metabolic disorder with the polypeptide of the present invention, and especially treatment and/or Prevent diabetes.
Another kind of antidiabetic (is for example relied on and not taken charge of for carnitine palmityl transferase I (CPT-I) inhibitor (etomoxir)), it can be used together to adjust blood with the FGF-21 polypeptides through modification in another embodiment of the present invention Sugar.Support does not take charge of irreversibly suppression carnitine palmityl transferase I, and this is required for fatty acid oxidation.Institute State and suppress that hyperglycaemia on an empty stomach can be reduced, because the product of fatty acid oxidation stimulates hepatic gluconeogenesis.Rely on Mo Sike Improve the insulin sensitivity of diabetes B patient.Hubinger et al., Hormone Metab.Res.24：115-18 (1992)。
Other known antidiabetic includes：It can be mentioned that insulin preparation (for example, extracting from the pancreas of ox and pig Animal insulin preparation；The human insulin's preparation synthesized using Escherichia coli, yeast with mode of inheritance；Insulin zinc；Milt Albumen insulin zinc；The fragment or derivative (for example, INS-1) of insulin, Macrulin)；Insulin sensitizer (example Such as, Pioglitazone (pioglitazone) or its salt (preferably hydrochloride), Rosiglitazone (rosiglitazone) or its salt are (excellent Select maleate), netoglitazone (Netoglitazone), RIVOGLITAZONE (Rivoglitazone) (CS-011), FK-614, Compound described in WO01/38325, prick in Ge Liezha (Tesaglitazar) (AZ-242), Roger (Ragaglitazar) (N, N-622), Mo Geliezha (Muraglitazar) (BMS-298585), according to lattice Liezong (Edaglitazone) (BM-13-1258), MAG reach gloomy (Metaglidasen) (MBX-102), Na Gelizha (Naveglitazar)(LY-519818)、MX-6054、LY-510929、AMG-131(T-131)、THR-0921)；α-glucoside Enzyme inhibitor is (for example, voglibose (voglibose), acarbose, Miglitol, emiglitate (emiglitate) Deng)；Biguanides is (for example, insoral (phenformin), melbine, buformin (buformin) or its salt are (for example, hydrochloric acid Salt, fumarate, succinate))；[sulfonylureas is (for example, orinase, glibenclamide for insulin secretagogue (glibenclamide), gliclazide, chlorpropamide, tolazamide, Acetohexamide, glyclopyramide (glyclopyramide), Glimepiride, Glipizide, glybuzole (glybuzole)), Repaglinide, Nateglinide (nateglinide), Mitiglinide (mitiglinide) or its calcium salt hydrate]；Inhibitors of dipeptidyl IV is (for example, dimension Ge Lieting (Vidagliptin) (LAF237), P32/98, sitagliptin (Sitagliptin) (MK-431), P93/01, PT- 100th, BMS-477118 (Saxagliptin) (BMS-477118), T-6666, TS-021))；The activators of β 3 (for example, AJ-9677)； GPR40 activators；Polypeptide (I) (glp I), (glp2) or the other diabetogenic peptide hormones of glucagon-like；GLP-1 by Body activator is [for example, GLP-1, GLP-1MR agent, N, N-2211, AC-2993 (exendin-4), BIM-51077, Aib (8,35) HGLP-1 (7,37) NH2、CJC-[131]]；Amylin (amylin) activator (for example, pramlintide (pramlintide))； Phosphotyrosine phosphatase inhibitor (for example, sodium vanadate)；Gluconeogenesis inhibitor is (for example, glycogen phosphorylase inhibitors, Portugal Grape sugar -6- inhibitors of phosphatases, glucagon antagonist)；SGLUT (sodium-glucose co-transporters body) inhibitor is (for example, T- 1095)；11beta-Hydroxysteroid dehydrogenase inhibitor (for example, BVT-3498)；Adiponectin (adiponectin) or its excitement Agent；IKK inhibitor (for example, AS-2868)；Improve the medicine of leptin (leptin) resistance；Somatostatin receptor agonist (compound described in WO01/25228, WO03/42204, W098/44921, W098/45285, W099/2273.5 etc.)；Portugal Sugared kinase activation agent (for example, R ° of -28-1675)；GIP (glucose-dependent-insulinotropic peptide) etc..
The FGF-21 polypeptides comprising alpha-non-natural amino acid combined individually or with other suitable components may be made as passing through Suck the aerosol composite (that is, it " can be atomized ") of administration.Aerosol composite can be placed into the acceptable propellant of pressurization In (such as dicholorodifluoromethane, propane, nitrogen).
Suitable for parenteral administration (for example, passing through intra-articular (in joint), intravenous, intramuscular, intracutaneous, intraperitoneal and skin Lower approach) composite include aqueous and non-aqueous isotonic aseptic injectable solution, it can contain antioxidant, buffer, suppression Microbial inoculum and make the composite solute isotonic with the blood of expected recipient；And aqueous and non-aqueous sterile suspensions, it can Including suspending agent, solubilizer, thickener, stabilizer and preservative.FGF-21 composite can be close in unit dose or multiple dose Seal in container (such as ampoule and bottle) and present.
Parenteral dispensing and Intravenous administration are preferred medication administration method.Specifically, natural amino acid homologue is had been used for Therapeutic agent dosing way (be including but not limited to generally used for EPO, it is GH, G-CSF, GM-CSF, IFN, interleukins, anti- The dosing way of body, FGF and/or any other pharmaceutically transferrin) provide this hair together with the composite used at present The preferred dosing way and composite of bright polypeptide.
In the context of the present invention, depending on the application, the dosage of administration patient is enough to produce in patient's body with the time Beneficial therapeutic response, or other suitable actives.Pass through effect and used non-natural amino of specific support or composite Activity, stability or the serum half-life of sour polypeptide and the symptom of patient and the body weight or surface area of patient to be treated are determined Dosage.Any harmful side effect that dosage size is also produced in particular patient body with the administration of specific support, composite etc. Presence, nature and extent determine.
It is determined that being intended in the treatment or prevention of disease (including but not limited to cancer, genetic disease, diabetes, AIDS etc.) During the effective dose of the carrier of administration or composite, doctor evaluates circulating plasma content, composite toxicity, progression of disease and/or (phase During pass) generation of anti-non-natural amino acid polypeptides antibody.
The dosage of such as kilogram patient of administration 70 is generally in the scope of the dosage equal to currently used therapeutic protein Interior, the scope can be adjusted for the change of compositions related activity or serum half-life.The present invention carrier or Pharmaceutical formulation can be by any of routine treatment come supplementary therapy condition, and the routine treatment includes antibody administration, epidemic disease Seedling administration, administration cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleotide analog, BRM etc..
On dispensing, composite of the invention is with by the LD-50 or ED-50 of relative allocation thing and/or to various concentration Non-natural amino acid polypeptides any side effect (including but not limited to when the body weight and holistic health for being related to patient) Observed result determined by speed administration.It can be realized and offerd medicine by single dose or divided dose.
If there is heating, felt cold or myalgia in the patient of experience composite transfusion, then he/her receives suitable dosage Aspirin (aspirin), brufen (ibuprofen), acetaminophen (acetaminophen) or other pain/heating control Pharmacy thing.30 minutes before it will infuse, with aspirin, acetaminophen or (including but is not limited to) diphenhydramine (diphenhydramine) to experience infusion reaction (for example heating, myalgia and feel cold) patient premedicate.Piperazine For pyridine (meperidine) even more serious felt cold and flesh for what can not rapidly be reacted to antipyretic and antihistaminic Meat pain.The seriousness of visual response and be slowed or shut off neutropenia.
The mankind FGF-21 polypeptides of the present invention can direct administration mammalian subject.By being generally used for individual introducing Any approach of FGF-21 polypeptides is offerd medicine.FGF-21 peptide compositions include being suitable to mouth according to an embodiment of the invention Clothes, per rectum, part, suction (including but not limited to by aerosol), buccal (including but not limited to sublingual), through the moon Road, it is parenteral (in including but not limited to subcutaneous, intramuscular, intracutaneous, intra-articular, pleura, intraperitoneal, intracerebral, intra-arterial or vein It is interior), the FGF-21 peptide compositions of local (that is, skin and mucomembranous surface, it includes tracheae surface) and transdermal administration, but It is any it is set in the case of most suitable approach by depending on the property and seriousness of treated symptom.Dispensing can be for locally or systemically Property dispensing.The composite of compound can be presented in unit dose or multiple dose sealing container (such as ampoule and bottle).This hair Bright FGF-21 polypeptides can be prepared as unit dosage injectable form (including but not limited to solution, suspension or emulsion) and doctor The mixture of pharmaceutically acceptable supporting agent.Also by continuous transfusion ((including but is not limited to) micropump can be used, for example, is permeated Pump), single bolus or sustained release storage tank formula composite come administration the present invention FGF-21 polypeptides.
Suitable for dispensing composite include the aqueous solution and non-aqueous solution (isotonic sterile solution), its can containing antioxidant, Buffer, bacteriostatic agent and make the isotonic solute of composite；And aqueous and non-aqueous sterile suspensions, its may include suspending agent, Solubilizer, thickener, stabilizer and preservative.Solution can be prepared by the aseptic powdery, particle and tablet of previously described species And suspension.
Freeze-drying is the technology for being generally used for presenting protein for going water removal from protein formulation of interest. Freeze-drying or it is lyophilized be make to be intended to dry material freezing first and then made a return journey by being distilled in vacuum environment deicing or The process of chilled solvent.It may include excipient to strengthen the stability in freezing dry process in lyophilized composite in advance And/or stability of the improvement lyophilized products in storage.Pikal, M.Biopharm.3 (9) 26-30 (1990) and Arakawa etc. People, Pharm.Res.8 (3)：285-291(1991).
Those skilled in the art is it is also known that the spray drying of medicine.For example, referring to Broadhead, J. etc. People, " The Spray Drying of Pharmaceuticals, " Drug Dev.Ind.Pharm, 18 (11 and 12), 1169- 1206(1992).In addition to small-molecule drug, also a variety of biomaterials are spray-dried and these biomaterials include：Enzyme, Serum, blood plasma, microorganism and yeast.Due to spray drying can by liquid pharmaceutical preparation with single step processes change into no dust or The fine powder of reunion, so spray drying is useful technology.Basic fundamental includes following four step：A) feedstock solution is atomized Into spray liquid；B) spray liquid-air contact；C) spray liquid is dried；And d) desciccate is separated with dry air.United States Patent (USP) No. 6,235,710 and No. 6,001,800 (it is to be incorporated herein by reference) description is made by being spray-dried Standby recombinant erythropoietin.
The medical composition and composite of the present invention can include pharmaceutically acceptable supporting agent, excipient or stabilizer. Pharmaceutically acceptable supporting agent is that part is determined by the particular composition of desire administration and for the ad hoc approach of administration composition It is fixed.Accordingly, there exist a variety of suitable composites of the medical composition of the present invention (including optional pharmaceutically acceptable load Agent, excipient or stabilizer).(referring for example to Remington ' s Pharmaceutical Sciences, the 17th edition, 1985).
Suitable supporting agent includes but is not limited to：Buffer, it contains succinate, phosphate, borate, HEPES, lemon Lemon hydrochlorate, histidine, imidazoles, acetate, bicarbonate and other organic acids；Antioxidant, including but not limited to Vitamin C Acid；The polypeptide of low molecular weight polypeptide, including but not limited to less than about 10 residues；Protein, including but not limited to serum Albumin, gelatin or immunoglobulin；Hydrophilic polymer, including but not limited to polyvinylpyrrolidone；Amino acid, including (but not limited to) glycine, glutamine, asparagine, arginine, histidine or histidine derivative, methionine, glutamic acid Or lysine；Monose, disaccharides and other carbohydrate, including but not limited to trehalose, sucrose, glucose, mannose or Dextrin；Chelating agent, including but not limited to EDTA and disodium ethylene diamine tetraacetate (edentate disodium)；Divalent metal Ion, including but not limited to zinc, cobalt or copper；Sugar alcohol, including but not limited to mannitol or D-sorbite；Into salt contend with from Son, including but not limited to sodium and sodium chloride；And/or nonionic surface active agent, including but not limited to TweenTM(including (but not limited to) Tween 80 (polysorbate80) and Tween 20 (polysorbate20)), PluronicsTMWith other pools Lip river niacin (pluronic acid) (including but not limited to Pluronic acid F68 (PLURONICS F87 (poloxamer )) or PEG 188).Suitable surfactant is for example including but not limited to poly- (ethylene oxide)-poly- (propylene oxide)-poly- (ethylene oxide) (that is, (PEO-PPO-PEO)) or poly- (propylene oxide)-poly- (ethylene oxide)-poly- (propylene oxide) (that is, (PPO- PEO-PPO)) or its combination based on polyethers.PEO-PPO-PEO and PPO-PEO-PPO is with trade name PluronicsTM、R- PluronicsTM、TetronicsTMAnd R-TetronicsTM(BASF Wyandotte Corp., Wyandotte, Mich.) exists Sell and be further described in U.S. Patent No. 4,820,352 on the market, the patent is in entirety by reference It is incorporated herein.Other ethylene/polypropylene block polymers can be suitable surfactant.Surfactant or surface-active The combination of agent can be used for stablizing Pegylation FGF-21 resisting one or more kinds of stress (including but not limited to by stirring Mix the stress of generation).Some above-mentioned substances can be referred to as " swelling agent (bulkingagent) ".Some are also known as " tension force Conditioning agent ".To reach product stability and antimicrobial efficiency, antibiotic antiseptic can be also applied；Suitable preservative includes (but not limiting In) benzylalcohol, benzalkonium chloride (bezalkonium chloride), metacresol, methyl p-hydroxybenzoate/P-hydroxybenzoic acid third Ester, cresols and phenol or its combination.
The FGF-21 polypeptides (including the FGF-21 polypeptides being connected with the water-soluble polymer such as PEG) of the present invention can also lead to Cross sustained release system or carry out administration as a part for sustained release system.Sustained-release composition includes (including but not limiting In) in the semipermeable polymer matrices of formed article (including but not limited to film or microcapsules) form.Sustained release base Matter include bio-compatible material, such as poly- (2-hydroxyethyl methacrylate) (Langer et al., J.Biomed.Mater.Res., 15：167-277(1981)；Langer, Chem.Tech., 12：98-105 (1982)), ethene Vinyl acetate (Langer et al., with above) or poly- D- (-) -3-hydroxybutyrate (EP 133,988), polylactide (poly- breast Acid) (U.S. Patent No. 3,773,919；EP 58,481), PGA (glycolic acid polymer), the poly- second of polylactide -co- Lactide (copolymer of lactic acid and glycolic), polyanhydride, copolymer (Sidman etc. of Pidolidone and γ-ethyl-L-glutamate ester People, Biopolymers, 22,547-556 (1983)), poe, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, Carboxylic acid, aliphatic acid, phosphatide, polysaccharide, nucleic acid, polyaminoacid, amino acid (such as phenylalanine, tyrosine, isoleucine), multinuclear Thuja acid, polyethylene propylene, polyvinylpyrrolidone and silicone.Sustained-release composition also includes the chemical combination that liposome is captured Thing.Liposome containing the compound is prepared by method known per se：DE 3,218,121；Epstein et al., Proc.Natl.Acad, Sci U.S.A., 82：3688-3692(1985)；Hwang et al., Proc.Natl Acad.Sci U.S.A., 77：4030-4034(1980)；EP 52,322；EP 36,676；U.S. Patent No. 4,619,794；EP 143, 949；U.S. Patent No. 5,021,234；Japanese patent application case 83-118008；U.S. Patent No. 4,485,045 and the 4th, No. 544,545；And EP 102,324.Cited all bibliography and patent are all hereby incorporated herein by In.
The FGF-21 polypeptides of liposome capture can be prepared by the method described in documents below：For example, DE 3,218, 121；Eppstein et al., Proc.Natl Acad.Sci.U.S.A., 82：3688-3692(1985)；Hwang et al., Proc.Natl.Acad.Sci.U.S.A., 77：4030-4034(1980)；EP 52,322；EP 36,676；U.S. Patent No. 4, No. 619,794；EP 143,949；U.S. Patent No. 5,021,234；Japanese patent application case 83-118008；U.S. Patent No. No. 4,485,045 and No. 4,544,545；And in EP 102,324.The composition and size of liposome to be well-known or Can rule of thumb it be readily determined by those skilled in the art.Some examples of liposome are described in (such as) Park JW et al., Proc.Natl.Acad.Sci.USA 92：1327-1331(1995)；Lasic D and Papahadjopoulos D (eds.)：MEDICAL APPLICATIONS OF LIPOSOMES(1998)；Drummond DC et al., Liposomal drug DeliverySystems for cancer therapy, Teicher B (eds.)：CANCER DRUG Discovery and Development(2002)；Park JW et al., Clin.Cancer Res.8：1172-1181(2002)；Nielsen UB etc. People, Biochim.Biophys.Acta 1591 (1-3)：109-118(2002)；Mamot C et al., Cancer Res.63： In 3154-3161 (2003).Cited all bibliography and patent are all incorporated herein by reference.
In the context of the present invention, it should be enough to produce beneficial reaction in individual with the time to the dosage of patient's administration. Generally, total medical effective dose of the FGF-21 polypeptides of the invention of every dose of parenteral administration in daily per kilogram weight in patients about To the microgram of per kilogram weight in patients about 100, or daily, about 0.05 milligram of per kilogram of body weight arrives per kilogram patient's body to 0.01 microgram Weigh about in the range of 1 milligram, but this is to be judged as foundation to treat.Administration frequency is also to be judged as foundation to treat, and can With more higher or lower than the frequency for ratifying the commercially available FGF-21 polypeptide products for the mankind.Generally, Pegylation of the invention FGF-21 polypeptides can pass through any of the above described dosing way administration.
In certain embodiments, modified FGF-21 polypeptides of the invention adjust the effect of antidiabetic.In the present invention Another embodiment in, modified FGF-21 polypeptides can be with the common administration of antidiabetic.In another implementation of the present invention Example in, can before being treated with antidiabetic administration modified FGF-21 polypeptides.In another embodiment of the present invention, may be used The administration modified FGF-21 polypeptides after being treated with antidiabetic.In another embodiment of the present invention, through modification FGF-21 polypeptides are and the common administration of melbine.In another embodiment of the present invention, presence or absence of insulin In the case of with the present invention modified FGF-21 polypeptides and melbine carries out therapeutic treatment can increase melbine regulation The ability of plasma glucose.In combination treatment, melbine with sulfonylureas, Alpha-glucosidase inhibitor, troglitazone and Insulin is used together.The combination increase insulin sensitivity of melbine and sulfonylureas simultaneously can reduce plasma glucose.Or, When maintaining glycemic control within the several months, melbine is more effective than Glipizide together with Repaglinide, and at least with it is excellent Hypoglycemic is equally effective.Melbine can improve glucose control together with troglitazone, and it is all more effective than independent any medicament. Inzucchi et al., New.Eng.J.Med.338：867-72(1998).In certain embodiments, FGF-21 polypeptides of the invention It is and the common administrations of Klotho beta.In certain embodiments, FGF-21 polypeptides of the invention be with including one or one with The common administrations of Klotho beta of upper non-naturally encoded amino acids.In certain embodiments, FGF-21 polypeptides of the invention be with Klotho beta and the common administration of antidiabetic.In certain embodiments, FGF-21 polypeptides of the invention are and anti-diabetic The common administration of medicine.In certain embodiments, FGF-21 polypeptides of the invention are combined with one or more kinds of agents Use：Taurine, alpha lipoic acid, Fructus Mori extract, chromium, glutamine, felwort, Sweet Broomwort Herb, tarragon extract and Herba Andrographitis. In certain embodiments, FGF-21 polypeptides of the invention are applied in combination with one or more kinds of agents：Insulin Preparation is (for example, the animal insulin preparation extracted from the pancreas of ox and pig；Closed using Escherichia coli, yeast with mode of inheritance Into human insulin's preparation；Insulin zinc；Protamine zinc insulin；The fragment or derivative of insulin are (for example, INS- 1), Macrulin)；Insulin sensitizer (for example, Pioglitazone or its salt (preferably hydrochloride), Rosiglitazone or its Salt (preferably maleate), netoglitazone, RIVOGLITAZONE (CS-011), the compound described in FK-614, WO01/38325, replace Pricked in Ge Liezha (AZ-242), Roger (N, N-622), Mo Geliezha (BMS-298585), according to lattice Liezong (BM-13-1258), beautiful Ge Dasen (MBX-102), Na Gelizha (LY-519818), MX-6054, LY-510929, AMG-131 (T-131), THR- 0921)；Alpha-glucosidase inhibitor (for example, voglibose, acarbose, Miglitol, emiglitate etc.)；Biguanides (example Such as, insoral, melbine, buformin or its salt (for example, hydrochloride, fumarate, succinate))；Insulin promotees to divide Secretin [sulfonylureas (for example, orinase, glibenclamide, gliclazide, chlorpropamide, tolazamide, Acetohexamide, Glyclopyramide, Glimepiride, Glipizide, glybuzole), Repaglinide, Nateglinide, Mitiglinide or its calcium salt hydration Thing]；Inhibitors of dipeptidyl IV (for example, vildagliptin (LAF237), P32/98, sitagliptin (MK-431), P93/01, PT-100, BMS-477118 (BMS-477118), T-6666, TS-021))；The activators of β 3 (for example, AJ-9677)；GPR40 excitements Agent；Polypeptide (I) (glp I), (glp2) or the other diabetogenic peptide hormones of glucagon-like；GLP-1 receptor stimulating agents [for example, GLP-1, GLP-1MR agent, N, N-2211, AC-2993 (exendin-4), BIM-51077, Aib (8,35) hGLP-1 (7,37) NH2、CJC-[131]]；Amylin agonist (for example, pramlintide)；Phosphotyrosine phosphatase inhibitor (for example, Sodium vanadate)；Gluconeogenesis inhibitor is (for example, glycogen phosphorylase inhibitors, G-6-Pase inhibitor, pancreas hyperglycaemia Plain antagonist)；SGLUT (sodium-glucose co-transporters body) inhibitor (for example, T-1095)；11beta-Hydroxysteroid dehydrogenase presses down Preparation (for example, BVT-3498)；Adiponectin or its activator；IKK inhibitor (for example, AS-2868)；Improve the medicine of leptin resistance Thing；Somatostatin receptor agonist (WO01/25228, WO03/42204, W098/44921, W098/45285, W099/2273.5 Compound Deng described in)；Activators of glucokinase (for example, R ° of -28-1675)；GIP (glucose dependency pancreotropic hormones Peptide).
In certain embodiments, polypeptide of the invention is used with following insulin reagents recombination：E.g., including (but It is not limited to) taurine, alpha lipoic acid, Fructus Mori extract, chromium, glutamine, felwort, Sweet Broomwort Herb, tarragon extract and punching Lotus.In another embodiment, the present invention can comprising one of isomalt, trehalose or D-MANNOSE or one with On to further enhance secretion or the activity of insulin.In another embodiment, also used in addition to another antidiabetic The polypeptide of insulin reinforcing agent and the present invention.
A kind of mode that can determine that the therapeutic efficiency of polypeptide and the combination treatment including polypeptide of the present invention is reduction patient HbAlc levels.In one embodiment, polypeptide of the invention reduction HbAlc levels, the HbAlc levels are with starting with through repairing Bimestrial HbAlc levels are compared, with starting first three treated with modified FGF-21 polypeptides before decorations FGF-21 polypeptide therapeutics The HbAlc levels of individual month turn to or change compared with baseline compared to change percentage be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%th, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%th, 40%, 45%, 50% or at least 50%.In another embodiment, the patient's that administration is also treated with antidiabetic Antidiabetic described in polypeptides for modulating of the present invention reduces the ability of blood glucose.In another embodiment, administration also uses anti-diabetic The polypeptide of the present invention reduction HbAlc levels of the patient of medicine treatment, the HbAlc levels are controlled with starting with modified FGF-21 polypeptides Trimestral HbAlc levels before bimestrial HbAlc levels are compared and start to be treated with modified FGF-21 polypeptides before treatment Turned to compared to change or be at least 1% compared to change percentage with baseline before baseline or treatment, 2%, 3%, 4%, 5%, 6%, 7%th, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%th, 40%, 45%, 50% or at least 50%.
In another embodiment, modified FGF-21 polypeptides of the invention regulation troglitazone reduces insulin requirements Ability.In another embodiment, modified FGF-21 polypeptides of the invention are in the patient that administration is just being treated with troglitazone The insulin requirements of the patient can further be reduced.In certain embodiments of the present invention, the song being applied in combination with the present invention Lattice row ketone can be used for postponing or preventing diabetes B.
In one embodiment of the invention, modified FGF-21 polypeptides are and the common administration of sulfonylureas.The present invention's In another embodiment, the administration modified FGF-21 polypeptides before with sulphonylurea therapy.In an alternative embodiment of the invention In, the administration modified FGF-21 polypeptides after with sulphonylurea therapy.In some embodiments of the invention, with therapeutic dose The adjustable serum glucose of modified FGF-21 polypeptides treatment.In another embodiment, FGF-21 polypeptides of the invention be with Adjust the Klotho beta of influence of the polypeptide to blood glucose administration together.In another embodiment, FGF-21 of the invention Polypeptide is the administration together with Klotho beta, and this is than being used alone the more effective reduction blood glucose of modified FGF-21 polypeptides.Another In individual embodiment, these changes are measured using HbAlc tests.In another embodiment, by the FGF-21 polypeptides of the present invention With Klotho beta administrations into the patient just treated with antidiabetic, this is than being used alone the more effective drop of antidiabetic Hypoglycemia.
XV. therapeutical uses of FGF-21 polypeptides of the invention
The FGF-21 polypeptides of the present invention are applied to treatment various disease conditions.
The FGF-21 polypeptides of the present invention can be used to suffer from adult-onset diabetes (NIDDM to treat：2 types), pancreas Island element dependent diabetes mellitus (1 type) and obesity, glucose clearance deficiency, hyperglycemia, hyperinsulinemia and can be by Any other disease of FGF-21 mediations or the mammal of symptom.Poor glucose tolerance may be defined as the sensitivity to glucose Sexual abnormality.The sugar (glucose) that hyperglycemia is defined as in blood is excessive.Hyperinsulinemia is defined as the pancreas in blood Island element is higher than normal level.Insulin resistance is defined as the shape of the subnormal biological respinse of insulin generation of normal amount State.For human individual, the ideal body weight that obesity may be defined as the set colony of weight ratio exceeds 20% (R.H.Williams, Textbook of Endocrinology, 1974, the 904-916 pages).
Diabetes are divided into two major classes according to clinical manifestation：That is, non-insulin-dependent or ripe breaking-out type, also referred to as 2 types； And insulin-dependent or juvenile onset, also referred to as 1 type.Clinically, the ripe breaking-out type diabetic of most of 2 types Person is more fat, generally occurs the clinical symptoms performance of disease after 40 years old.By contrast, although type 1 diabetes can be in office What occurs at the age, but 1 type juvenile onset patient is not overweight for its age and height, young (usual 30 years old Disease is broken out rapidly when before).
Diabetes are a kind of mankind's metabolic disorders, and its incidence of disease in total group is about 1%, wherein four in these colonies / mono- is 1 type (Foster, D.W., Harrison ' s Principles of Internal Medicine, the 114th chapter, the 661-678 pages, the 10th edition, McGraw-Hill, New York).The disease manifests themselves as a series of generations by steroid-induced Thank to exception, it, which is ultimately resulted in, is related to the serious long-term and weak of some tracts (including eyes, kidney, nerve and blood vessel) Complication.On pathology, the feature of the disease is the lesion of basilar memebrane, and this can be confirmed under an electron microscope.
Adult-onset diabetes (NIDDM：2 types) it is that one kind is characterized as high circulation blood glucose, insulin and cortex class The debilitating disease of sterol levels.The incidence of disease of diabetes B is higher and is still rising, and is just becoming dead in worldwide Main cause (Amos et al., the Diabetic Med.14 of rate, the incidence of disease and health care expenditure：S1-85,1997).
Not yet it is fully understood by the cause of disease of diabetes B.Although the feature of diabetes B is that insulin can be used, but still is deposited Produced in excessive glucose, and circulating-glucose levels are still too high because of glucose clearance deficiency.Think target tissue pair The resistance of insulin action reduces (" failure of β cells ") with insulin secretion all to be occurred.The main pancreas islet of glucose homeostasis Plain reactivity tissue is liver, wherein insulin stimulating Glycogen synthesis and suppresses gluconeogenesis；Muscle, wherein insulin stimulating Portugal Grape Sugar intake, and glycogen stimulates glucose uptake and suppresses lipolysis.Therefore, because the Portugal in diabetes symptom, blood Grape sugar level is raised, and hyperglycaemia extends, and this indicates the symptom by blood vessel and neurotrosis is caused.
It is currently, there are pharmacological method (Scheen et al., the Diabetes Care, 22 of a variety for the treatment of diabetes Bs (9)：1568-1577,1999).It is worked by different binding modes：1) sulfonylureas substantially stimulates insulin secretion；2) Biguanides (melbine) is acted as by promoting glucose utilization, reducing hepatic glucose generation and reducing the output of intestines glucose With；3) Alpha-glucosidase inhibitor (acarbose, Miglitol) slows down carbohydrate digestion and therefore reduced from internal organ Middle absorption carbohydrate, and reduce hyperglycaemia after meals；4) thiazolidinedione (troglitazone) enhancing insulin action, therefore Promote the glucose utilization in surrounding tissue；Utilized with 5) insulin stimulating tissue glucose and suppress hepatic glucose output.On Stating pharmacological method can individually utilize or be utilized in combination treatment form.However, each method all has its limitation and ill-effect.
Obesity is a kind of popular chronic disease of modern society, and it is not only relevant with social discrimination, and Also with service life reduction and including bad mental development, dermatological conditions (such as infecting), varication, tolerance of exercise not Foot, diabetes, insulin resistance, hypertension, hypercholesterolemia are relevant with numerous medical problems including coronary heart disease. Rissanen et al., British Medical Journal, 301：835-837(1990).
The existing therapy of obesity includes：Standard diet and motion；Very low calorie diet；Behavior therapy；It is related to appetite suppression Preparation, the medicine (thermogenic drug) for increasing energy expenditure, the medicinal treatment of food absorption inhibitor；Mechanical device, Such as facing (jaw wiring), (waist cord) and balloon with a tight waist；And operation.Jung and Chong, ClinicalEndocrinology, 35：11-20(1991)；Bray, Am.J.Clin.Nutr., 55：538S-544S(1992).
It is potential in view of high popular and as described above associated serious consequence of the obesity in human society Can all have far-reaching beneficial effect to its health suitable for any curative drug of the body weight of the fat personnel of reduction.Affiliated neck Need the total weight of obese individuals will be reduced into its ideal body weight in the case of without notable adverse side effect in domain and incite somebody to action Obese individuals are contributed to maintain the medicine of the weight levels of reduction.
Accordingly, it is desirable to provide a kind of be applied to make the weight recoveries of obese individuals to the treatment side of normal ideal body weight Case.Additionally, it is desirable to provide a kind of obesity therapy, it causes long-term to maintain the body weight of reduction.
In experimental animal and the mankind, obesity and insulin resistance and diabetes height correlation.In fact, obesity and Insulin resistance (latter of which is generally along with hyperinsulinemia or hyperglycemia or both) is the mark of diabetes B. In addition, diabetes B makes the risk of coronary artery disease increase to twice to four times.Although on these serious health problems Research reaches many decades, but obesity and the cause of disease of insulin resistance still belong to unknown.
1 type diabetic person characteristically shows extremely low or immeasurablel plasma insulin and elevated pancreas hyperglycaemia Element.Regardless of the definite cause of disease, most of 1 type patients have the circulating antibody for its own pancreatic cell, including pancreas islet Plain antibody, cytoplasmic antibody and enzyme glutamic acid decarboxylase antibody for islet cells.Specificity (produces pancreas for β cells Island element cell) immune response cause type 1 diabetes.This species specificity obtains above-mentioned clinical picture and supported, because β cells point Secrete insulin, and α cells secrete glucagons.
The Current treatment protocols of type 1 diabetes include adjustment diet to minimize caused by natural insulin lacks Hyperglycaemia, and natural insulin shortage is the result of β cell damages.Also adjust diet to resist hormone on insulin administration Hypoglycemia effect.Regardless of form of therapy, all 1 type diabetic persons are required for parenteral insulin administration, therefore are referred to as " insulin-dependent " diabetes.
Accordingly, it would be desirable to than the effective diabetes B therapy of the few side effect of pharmaceutical methods can be used.In addition, effectively Insulin replacement therapy be equally applicable to treat type 1 diabetes.The present invention provides a kind of pharmacological treatments, and it stimulates glucose Absorb and strengthen the insulin sensitivity in surrounding tissue, and with the secondary work fewer than current diabetes B therapeutic scheme With.In addition, the present invention provides a kind of replacement therapy of type 1 diabetes.In addition, the present invention is because by faster and more effective Portugal Grape sugar utilize increase energy expenditure and suitable for treating obesity.
The present invention provides a kind for the treatment of and shows type 1 diabetes, diabetes B, obesity, insulin resistance, hyperinsulinism The method of the mammal of one or more kinds of illnesss in mass formed by blood stasis, poor glucose tolerance or hyperglycemia, its include to Need the FGF-21 polypeptides of the invention of the mammal administration therapeutically effective amount of the treatment.
The treatment method can be enough to reach that below at least one changes in the mammal：Body fat storage reduction, Insulin resistance reduction, hyperinsulinemia mitigate, glucose tolerance increase and hyperglycaemia are reduced.
On the other hand, the present invention relates to the method that one kind treats domestic animal (including but not limited to ox, pig, sheep, horse etc.), its FGF-21 comprising administration effective dose or its variant, to reduce body fat storage.What body fat was stored in domestic animal body is long-term or permanent Property reduce substantially can the mankind be produced with significant economic interests, this is especially because animal supplies the major part of human diet；And And the form that animal tallow fatty can be piled up in human body again is terminated.
FGF2 1 (FGF-21) can be used for reducing the morbidity and mortality relevant with critical patient. Referring to U.S. Patent Publication case the 20050176631st, it is to be incorporated herein in entirety by reference.Long-term is needed to add The critical patient nursed by force has higher mortality risk and sizable death rate.Receive patient and enter intensivecare unit (intensive care unit；ICU common cause) is and infectivity attacks (septicemia) and such as pancreatitis, part The non-infectious causes for pathological such as ischemic, multiple trauma and tissue damage, hemorrhagic shock and immune-mediated organ damage has The systemic inflammation reaction syndrome (SIRS) of pass.The present invention is also covered by a kind of reduction and suffered from and infectious invasion and attack and non-infection Property causes for pathological it is relevant systemic inflammation reaction syndrome (SIRS) critical patient the death rate and the incidence of disease method, its Include the FGF-21 to critical patient administration therapeutically effective amount.Being related to the example of SIRS symptom includes septicemia, pancreatitis, office Portion's ischemic, multiple trauma and tissue damage, hemorrhagic shock, immune-mediated organ damage, acute respiratory distress syndrome (ARDS), shock, kidney failure and multiple organ dysfunction syndrome (MODS).The present invention is also covered by a kind of reduction, and to suffer from breathing embarrassed The death rate of urgent critical patient and the method for the incidence of disease
SIRS common complication is the development of organ system dysfunction, including acute respiratory distress syndrome (ARDS), shock, kidney failure and multiple organ dysfunction syndrome (MODS), it all expands the risk of unfavorable result.To the greatest extent The many experts of pipe think that some type of nutritional support is beneficial but good due to lacking to helping critical patient to recover metabolic stability The randomized clinical trial controlled well, the benefit of the support and detailed description still suffer from dispute.
Because common hyperglycaemia and insulin resistance in the critical patient for give nutritional support, some ICU administrations Insulin treats the excessive hyperglycaemia of the critical patient of feed.In fact, research recently confirms to use exogenous insulin by blood The level that sugar maintains not higher than 110 milligrams/deciliter can reduce in operation intensivecare unit the incidence of disease of critical patient and dead Rate is died, whether there is diabetic history (Van den Berghe et al., N Engl J Med., 345 (19) but regardless of it：1359, 2001)。
The present invention covers a kind of method for the death rate and the incidence of disease for reducing these critical patients by administration FGF-21. The critical patient that the present invention is covered typically undergoes unstable hypermetabolism state.This unstable metabolism state is due to base The change of matter metabolism, it can cause the relative shortage of some nutrients.The oxidation of fat and muscle generally increases.
Administration FGF-21 can reduce the critical patient of the death rate and the risk of the incidence of disease, and to be preferably experience systemic inflammation anti- Answer the patient of syndrome or respiratory distress.Incidence of disease reduction means that reduction critical patient will suffer from Other diseases, symptom or disease The possibility of shape or the seriousness of reduction Other diseases, symptom or symptom.For example, incidence of disease reduction may correspond to bacteremia Or the incidence reduction of septicemia or the complication related to multiple organ failure.
Systemic inflammation reaction syndrome (SIRS) describe the inflammatory process related to a large amount of clinical conditions and including (but It is not limited to) one or more of following clinical manifestation performance：(1) body temperature is higher than 38 DEG C or less than 36 DEG C；(2) heart rate is higher than every point Clock 90 times；(3) it is short of breath, shows as more than 20 times per minute breathings of respiratory rate；Or hyperventilation, such as PaCo2It is less than Shown in 32mm Hg；And the change of (4) white blood cell count(WBC), for example count and be more than 12,000/cu mm, count and be less than 4,000/cu Mm, or there is more than 10% immature neutrophil.These physiological changes should be represented in the absence of abnormal its With baseline in the case of its known reason (such as chemotherapy, inductivity Neutropenia and leukopenia) The acute change compared.
Septicemia is defined as the SIRS caused by infection.SIRS non-infectious pathogenesis may include pancreatitis, office It is portion's ischemic, multiple trauma and tissue damage (including but is not limited to compression injury or serious burn), hemorrhagic shock, immune The organ damage of mediation, and the external source throwing of amboceptor is estimated in inflammatory process such as TNF and other cell factors With.
Septic shock and multiple organ dysfunction are the main causes of morbidity and mortality in ICU environment.Septicemia With a variety of host defense mechanisms (including cytokine network, leucocyte and complement cascade and blood coagulation/fibrin including endothelium Fibrinolytic system) mediated about and by it.It is relevant with the Fibrin deposits in the microvasculature of multiple organs The consumption coagulopathy of disseminated intravascular coagulation (DIC) and other degree is the performance of septicemia/septic shock.Host prevents Imperial reaction be to the downstream effects of target organ the developing important amboceptor of multiple organ dysfunction syndrome (MODS) and Cause the poor prognosis of patient concurrently suffered a shock with septicemia, Severe sepsis and septicemia.
Respiratory distress represents that patient has dyspneic symptom because of some type of pulmonary dysfunction.These patients are led to Often show different degrees of hypoxemia, it is likely difficult to or may be not difficult to control by using supplemental oxygen treatment.Breathing is embarrassed Compel to occur in the patient due to direct injury of lungs with impaired PFT, or may be due to indirect injury of lungs (for example In the situation of systemic process) and occur.Additionally, there are a variety of predisposed illnesss substantially increases risk, as there is example As the secondary cause such as Chronic Alcohol abuse, chronic lung disease and low serum pH.
Some direct injury of lungs reasons include pneumonia, gastric content, and suction, contusion of lung, fat embolism, nearly drowned, suction are damaged by mistake Wound, mountain sickness (high altitude) and the reperfusion pulmonary edema after lung transplantation or pulmonary embolectomy.Some indirect lungs are damaged Hinder reason including septicemia, with shock and the severe trauma of many donors blood transfusion；Extracorporal circulatory system (cardiopulmonarybypass), the infusion of overdose, acute pancreatitis and blood product.
One class causes the lung conditions of respiratory distress relevant with the syndrome referred to as pulmonary heart disease (Cor Pulmonale). These illnesss are relevant with chronic hypoxemia, so as to cause pressure rise in pulmonary circulation (being referred to as pulmonary hypertension).Subsequent lung is high The live load of pressure increase right ventricle, therefore cause it to increase or loose.Pulmonary heart disease is usually expressed as right heart failure, right heart failure Be defined as right ventricular pressure continue to increase and clinical evidence show return to the right heart vein reduce.
Chronic obstructive pulmonary disease (COPD) (it includes pulmonary emphysema and chronic bronchitis) also result in respiratory distress and with Air flow obstruction is characterized.COPD is the fourth-largest cause of death and takes away the life of more than 100,000 every year.
Acute respiratory distress syndrome (ARDS) be usually progressive and be characterized with different phase.The syndrome Normally behave as the rapid onset of respiratory failure in the patient of the risks and assumptions with the symptom.It is difficult to by using supplemental oxygen The arterial blood oxygen deficiency for treating to control is characteristic feature.Alveolar filling may be present, consolidates and the lung present in related lung area Atelectasis；However, non-relevant areas may have parenchyma inflammation.The syndrome can develop into fibrosing alveolitis with holding Continuous hypoxemia, increases the dead space of alveolar and further reduces lung compliance.Also it can produce and be damaged by lung capillaries bed Pulmonary hypertension caused by wound.
The seriousness of clinical lung injury is different.The present invention covers (is defined as artery oxygen with lower severity hypoxemia Pressure and the ratio of suction oxygen part are 300 or less than 300) patient with (being defined as arterial oxygen with more serious hypoxemia Partial pressure and the ratio of suction oxygen part are 200 or less than 200) patient.Patient with 300 or less than 300 ratio is general It is classified as with ALI, and the patient with 200 or less than 200 ratio is classified as with acute respiratory distress Syndrome.
The feature of the acute stage of ALI for due to the vascular permeability of alveolar capillary barrier increase make richness Edematous fluid containing protein is flowed into air space.The forfeiture for the epithelial integrity that permeability changes is set to cause alveolar Drowning (alveolar flooding), destruction normal fluid conveying (it influences removal of the edematous fluid from alveolar space), reduces table The generation and renewal of face activating agent, cause septic shock, and cause fibrosis in the patient with bacterial pneumonia.Lose Mass formed by blood stasis is relevant with the highest risk for developing into ALI.
In the symptom such as septicemia, if there is hypermetabolism, then breaks down proteins should accelerate to maintain sugar Former heteroplasia and discharge protein synthesis increase needed for amino acid.Hyperglycemia can be presented and can be presented highly concentrated in serum The triglycerides of degree and other lipids.
The patient being damaged for respiratory function, hypermetabolism can influence carbon dioxide to produce the ratio consumed with oxygen.This One ratio is referred to as respiratory quotient (R/Q) and in normal individual between about 0.85 and about 0.90.Excess fat metabolism tool There is reduction R/Q trend, and excessive glucose metabolism raises R/Q.Respiratory distress patient is generally difficult to eliminate carbon dioxide simultaneously And therefore there is abnormal high respiratory quotient.
The critical patient generally also experience markses that the present invention is covered are stopped for the of short duration lower mediation heat of most cells product The specific stress reaction of gram protein upregulation.In addition, this stress reaction is related to such as hyperglycemic factor, growth hormone, cortisol (Cortisol) and the hormone such as pro-inflammatory cytokine and anti-inflammatory cytokine activation.Protected although this stress reaction seems to have Shield is acted on, but the reaction also produces other metabolism unstability in these critical patients.For example, these specific hormones Activation cause serum glucose to raise, it causes hyperglycemia.In addition, the damage to heart and other organs can be because of adrenal gland Element can be stimulated and aggravated.Furthermore, it is possible to change in the presence of to thyroid, it may have on metabolic activity significantly affects.
FGF-21 average magnitude can change and especially should be on the basis of the recommendation of qualified physicians and prescription.FGF-21 is really The amount of cutting preferably is influenceed by for example following factor：The exact type for the symptom treated, the health status and group for treating patient Other compositions in compound.The present invention also provides another activating agent of administration therapeutically effective amount.Those skilled in the art Given amount can be readily determined according to the therapy using FGF-21.
The medical composition of the present invention can be manufactured in a usual manner.
Example
Following instance is provided to illustrate the present invention that (but being not intended to limit) is advocated.
Example 1
This example describes many groups of possible marks for selecting the site that non-naturally encoded amino acids are incorporated in FGF-21 One group in standard.
Fig. 1 is shown as using carrier NTI (Invitrogen；Carlsbad, CA) determined FGF-21 (protein deposit Numbering BCO18404) sequence homology between FGF-19 (protein deposit numbering BAA75500).Between both molecules The amino acid marked with an asterisk is similar.The amino acid underlined between both polypeptides is identical.By using non-day Right coded amino acid replaces natural coded amino acid and produces several different FGF-21 polypeptides.Each polypeptide has a kind of warp pair The amino acid (being indicated in Fig. 1 with rectangle) of acetyl phenyl alanine substitution.Produced polypeptide lacks shown in Fig. 1 and Fig. 3 Targeting sequencing, and carry out His marks by 6 histidine residues at N-terminal.SEQ ID NO.：1 is not have targeting sequencing Mankind FGF-21 (p-shaped formula) 181 amino acid sequence.SEQID NO.：2 be without targeting sequencing and in N-terminal The sequence of mankind FGF-21 (p-shaped formula) with His labels.Produced each polypeptide is in SEQ ID NO：1 lower column position 10, 52nd, there is non-naturally encoded amino acids substitution at a position in 77,117,126,131 and 162.
Fig. 2 is shown obtained from Protein Data Bank (Protein Data Bank；PDB) (Bernstein et al., J.Mol.Biol1997, page 112,535) (1PWA) and use PyMOL softwares (DeLano Scientific；Palo Alto, CA) mark mankind FGF-19 structure.The present invention FGF-21 polypeptides in, corresponding to FGF-19 V34, L79, G104, S144, K155, L160 and S196 amino acid are by replacing to acetyl phenyl alanine.Dotted line is represented in prototype structure The region not parsed.
Fig. 3 is shown as using carrier NTI (Invitrogen；Carlsbad, CA) determined FGF-21 (protein deposit Numbering BCO18404) sequence homology between FGF-2 (protein deposit numbering BAA75500).Used between both molecules The amino acid that asterisk is indicated is similar.The amino acid underlined between both polypeptides is identical.Conduct is shown in Fig. 1 7 amino acid in substitution site also are located in Fig. 3 rectangle.
Fig. 4 is shown obtained from PDB (1CVS) and using PyMOL softwares (DeLano Scientific；Palo Alto, CA) The structure of mankind's FGF-2 structures of mark.Gray structure is mankind's FGF receptor 1 (FGFR1), and black structures are the mankind FGF2.Plotnikov, AN et al., Cell.1999 Septembers 3 days；98(5)：The FGF2's that 641-50 descriptions are combined with FGF receptor Crystal structure.In the FGF-21 polypeptides of the present invention, corresponding to FGF-2 F21, K62, K86, V125, K134, T139 amino Acid is by replacing to acetyl phenyl alanine.Dotted line represents the region not parsed in prototype structure.
Another group of standard for being used for the preferred sites that selection is incorporated to non-naturally encoded amino acids is as follows.Egg is come from using 10 kinds The crystal structure of white matter database simulates FGF-21 structure：1PWA (mankind FGF-19)；1IJT (mankind FGF-4)；1NUN (mankind FGF10-FGF acceptor 2b compounds)；1G82 (has FGF receptor and the mankind FGF-9 dimers of heparin)；1IHK (people Class FGF-9)；1BAR (ox FGF-1)；1QQK (rat FGF-7)；1K5U (mankind FGF-1)；1FQ9 (has FGF receptor 1 and liver The mankind FGF-2 of element)；With 2FDB (mankind FGF-8b with FGF receptor 2c).The coordinate of these structures is available from albumen prime number According to storehouse (PDB) (Bernstein et al., J.Mol.Biol.1997,112, page 535).The comparison of crystal structure in core texture It is all extremely similar to represent it.However it has been found that the N-terminal and C-terminal of these FGF molecules are extremely different, and therefore can not Simulate the end.The simulation identifies two residues, i.e. Y22 and Y104, and its is highly conserved and is combined with acceptor relevant. Also two potential heparin-binding sites are identified, it is related to R36 and E37.Differentiated amino acid position is combined on acceptor Correspond to SEQ ID NO with Heparin-binding residue：1.
Therefore, following residue is differentiated：1) its without interference with and FGF receptor or heparin combination, 2) it is not present in egg The inside of white matter, and 3) its by the substantially uniform region of crystal structure.In certain embodiments, one or one with Upper non-naturally encoded amino acids are incorporated at one or more positions in (but not limited to) FGF-21 following position： SEQ ID NO：1 87,77,83,72,69,79,91,96,108 and 110 or in SEQ ID NO：Corresponding amino acid in 2-7. In certain embodiments, one or more non-naturally encoded amino acids are in (but not limited to) FGF-21 following position One or more positions at be incorporated to：SEQ ID NO：1 87,77,83,72 or in SEQ ID NO：It is corresponding in 2-7 Amino acid.In certain embodiments, one or more non-naturally encoded amino acids be (but not limited to) FGF-21 with It is incorporated at one or more positions in lower position：SEQ ID NO：1 69,79,91,96,108 and 110 or in SEQ ID NO：Corresponding amino acid in 2-7.
Each FGF-21 positions for introducing non-naturally encoded amino acids are assessed using following standard：(a) according to structure Analysis, residue should not interfere with and FGF-21 acceptors combination；(b) residue should not be influenceed by alanine or homolog-scanning mutagenesis； (c) residue should be that surface exposes and show the minimum Van der Waals force (van der Waals) or hydrogen bonding with neighboring residues Interaction；(d) residue should be lacked or variable in FGF-21 variants；(e) residue replaces through non-naturally encoded amino acids After can produce conservative changes；And residue may be present in height pliability region or structure in rigid region (f).
Other or different crystal structure (such as FGF-23 and/or FGF-19 knots of FGF family members can also be used Structure) select to be incorporated to the sites of one or more non-naturally encoded amino acids in FGF-21.For example, mankind FGF-19 The crystal structure and/or mankind FGF-19 (PDB ID 2P23) and/or mankind FGF-23 (PDB ID of (PDB ID 2P23) Crystal structure 2P39) can provide the other information in the site on being incorporated to non-naturally encoded amino acids in selection FGF-21.This A little sites can be in the different zones of protein, and the region includes but is not limited to N-terminal and C-terminal, acceptor are combined and liver Plain calmodulin binding domain CaM.In addition, right using Cx programs (Pintar et al., (2002) Bioinformatics, the 18, the 980th page) FGF-21 molecules further calculate assessing the degree that each protein atomic is protruded.
In certain embodiments, one or more non-naturally encoded amino acids are incorporated in FGF-21 following position In one or more positions：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14, 15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、 40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、 65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、 90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、 111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、 130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、 149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、 168th, 169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl of protein End) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).In certain embodiments, one or one with Upper non-naturally encoded amino acids are incorporated in one or more positions in FGF-21 following position：10、52、117、126、 131st, 162,87,77,83,72,69,79,91,96,108 and 110 (SEQ ID NO：1 or in SEQ ID NO：It is corresponding in 2-7 Amino acid).In certain embodiments, one or more non-naturally encoded amino acids are incorporated in FGF-21 following position In one or more positions：10th, 52,77,117,126,131 and 162 (SEQ ID NO：1 or in SEQ ID NO：In 2-7 Corresponding amino acid).In certain embodiments, one or more non-naturally encoded amino acids be incorporated to FGF-21 with bottom In one or more positions put：87、77、83、72(SEQ ID NO：1 or in SEQ ID NO：Corresponding ammonia in 2-7 Base acid).In certain embodiments, one or more non-naturally encoded amino acids are incorporated to one in FGF-21 following position In individual or more than one position：69th, 79,91,96,108 and 110 (SEQ ID NO：1 or in SEQ ID NO：It is corresponding in 2-7 Amino acid).
Example 2
This Examples detail includes clone and expression of the FGF-21 polypeptides of non-naturally encoded amino acids in Escherichia coli.This Example also describes a kind of method for the bioactivity for analyzing modified FGF-21 polypeptides.
Those skilled in the art becomes known for the method for cloning FGF-21.FGF-21 polypeptide and polynucleotide sequence It is to be specified in U.S. Patent No. 6,716,626 with FGF-21 is cloned into host cell；U.S. Patent Publication case the 2005/th No. 0176631, No. 2005/0037457, No. 2004/0185494, No. 2004/0259780, No. 2002/0164713 With No. 2001/0012628；WO 01/36640；WO 03/011213；WO 03/059270；WO 04/110472；WO 05/ 061712；WO 05/072769；WO 05/091944；WO 05/113606；WO06/028595；WO 06/028714；WO 06/ 050247；WO 06/065582；In WO 06/078463, the document is to be incorporated herein in entirety by reference.
The cDNA of FGF-21 of the coding without targeting sequencing p-shaped formula is with SEQ ID NO：8 displayings.The polypeptide with SEQ ID NO：1 displaying.
The cDNA of FGF-21 of the coding without the targeting sequencing p-shaped formula marked through His is with SEQ ID NO：9 exhibitions Show.The polypeptide is with SEQ ID NO：2 displayings.
The cDNA of FGF-21 of the coding with the targeting sequencing containing 3 leucines together p-shaped formula is with SEQ IDNO： 10 displayings.The polypeptide is with SEQ ID NO：3 displayings.
The cDNA of FGF-21 of the coding with the targeting sequencing containing 2 leucines together p-shaped formula is with SEQ IDNO： 11 displayings.The polypeptide is with SEQ ID NO：4 displayings.
The cDNA of FGF-21 of the coding without targeting sequencing L-shaped formula is with SEQ ID NO：12 displayings.The polypeptide With SEQ ID NO：5 displayings.
The cDNA of FGF-21 of the coding with the targeting sequencing containing 3 leucines together L-shaped formula is with SEQ IDNO： 13 displayings.The polypeptide is with SEQ ID NO：6 displayings.
The cDNA of FGF-21 of the coding with the targeting sequencing containing 2 leucines together L-shaped formula is with SEQ IDNO： 14 displayings.The polypeptide is with SEQ ID NO：7 displayings.
Use the introduced translation system comprising orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synzyme (O-RS) Unite to express the FGF-21 containing non-naturally encoded amino acids.O-RS preferentially makes O-tRNA aminoacyls using non-naturally encoded amino acids Base.Then, the translation system responds encoded selection codon and inserts non-naturally encoded amino acids in FGF-21. Suitable O-RS and O-tRNA sequences are to be described in entitled " Compositions ofAminoacyl-tRNA Synthetase And Uses the Thereof " (E9 of WO 2006/068802；SEQ ID NO：And entitled " Compositions of 15) TRNA and Uses the Thereof " (F13 of WO 2007/021297；SEQ IDNO：16) in, the document is to draw in full Mode is incorporated herein.
Table 2：O-RS and O-tRNA sequences
SEQ ID NO：17 Methanococcus jannaschii mtRNACUA Tyr tRNA
SEQ ID NO：18 HLAD03；The amber suppressor tRNA of optimization tRNA
SEQ ID NO：19 HL325A；The AGGA frameshift suppressors tRNA of optimization tRNA
SEQ ID NO：20 For being incorporated to the aminoacyl tRNA synthetase to azido-L-phenylalanine p-Az-PheRS(6) RS
SEQ ID NO：21 For being incorporated to the aminoacyl tRNA synthetase to benzoyl-L-phenylalanine p-BpaRS(1) RS
SEQ ID NO：22 Aminoacyl tRNA synthetase for being incorporated to propargyl-phenylalanine Propargyl-PheRS RS
SEQ ID NO：23 Aminoacyl tRNA synthetase for being incorporated to propargyl-phenylalanine Propargyl PheRS RS
SEQ ID NO：24 Aminoacyl tRNA synthetase for being incorporated to propargyl-phenylalanine Propargyl-PheRS RS
SEQ ID NO：25 For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS(1) RS
SEQ ID NO：26 For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS(3) RS
SEQ ID NO：27 For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS(4) RS
SEQ ID NO：28 For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS(2) RS
SEQ ID NO：29 For being incorporated to the aminoacyl tRNA synthetase to acetyl-phenylalanine (LW1) RS
SEQ ID NO：30 For being incorporated to the aminoacyl tRNA synthetase to acetyl-phenylalanine (LW5) RS
SEQ ID NO：31 For being incorporated to the aminoacyl tRNA synthetase to acetyl-phenylalanine (LW6) RS
SEQ ID NO：32 For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine (AzPheRS-5) RS
SEQ ID NO：33 For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine (AzPheRS-6) RS
With containing modified FGF-21 gene and orthogonal aminoacyl tRNA synzyme/tRNA to (being compiled to required non-natural Code amino acid tool specificity) plasmid conversion Escherichia coli allow non-naturally encoded amino acids locus specificity being incorporated to FGF- In 21 polypeptides.
Expanded using standard scheme according to cDNA synthetic reactions by PCR obtained from healthy human liver polyA+mRNA (Biochain) wild-type mature FGF-21, and be cloned into pET30 (NcoI-BamHI).After sequence confirmation, in source Will under the composition control of the synthetic promoter (Miller, J.H., Gene, 1986) of E. coli lipoproteins promoter sequence FGF-21 including N-terminal HHHHHHSGG sequences is subcloned into containing the amber suppressor junket ammonia from Methanococcus jannaschii Acyl group tRNATyr/CUA(Mj tRNATyr/CUA) suppression carrier in, and Escherichia coli GlnRS promoters control under be subcloned Into the suppression carrier containing orthogonal tyrosyl- tRNA synzyme (MjTyxRS).FGF-21 expression is in T7 promoters Under control.Quickly change mutation scheme (Stratagene using standard；La Jolla, California) introduce amber mutation. All constructs all pass through sequence verification.
Suppressed with to acetyl-phenylalanine (pAcF)
Plasmid (pVK3-HisFGF21) is transformed into the W3110 B2 bacterial strains of Escherichia coli, wherein the table of T7 polymerases Up to being under the control of arabinose inducible promoter.Bacterial cultures overnight is diluted to 1: 100 and trained containing 2X YT Support in the vibration flask of base and grow into OD at 37 DEG C600It is about 0.8.By adding arabinose (0.2% ultimate density) To induce protein expression, and it is 4mM to add to acetyl-phenylalanine (pAcF) to ultimate density.By culture 37 Cultivated 4 hours at DEG C.Make cell glomeration and be resuspended in B-PER dissolvings buffer solution (Pierce) 100 μ l/OD/ml+10 μ Cultivated 30 minutes in g/ml deoxyribonucleases and at 37 DEG C.By centrifuging removal cellular material and removing supernatant Liquid.Bead is resuspended in equivalent SDS-PAGE protein load buffer solutions.All samples are all loaded into MES and On DTT 4-12%PAGE gels.The purification process of FGF-21 known to those skilled in the art, and methods described by SDS-PAGE, Western blot analysis or electron spray-ionization ion trap mass spectrometry etc. is confirmed.
Suppression at expression and 7 amber sites of the N-terminal through the His FGF-21 marked is shown by Fig. 5.Use arrow mark Bright FGF-21 polypeptides.Fig. 5 shows B-PER bead samples：1 road：Mark；2 roads：VK3-FGF21 pre-induced, supernatant；3 roads： VK3-FGF21 pre-induced, bead；4 roads：VK3-FGF210.2% arabinoses, supernatant；5 roads：VK3-FGF210.2% I Uncle's sugar, bead；6 roads：VK3-FGF21-pAcF-L10,0.2% arabinose；7 roads：VK3-FGF21-pAcF-L52,0.2% Ah Draw uncle's sugar；8 roads：VK3-FGF21-pAcF-R77,0.2% arabinose；9 roads：VK3-FGF21-pAcF-HI 17,0.2% Ah Draw uncle's sugar；10 roads：VK3-FGF21-pAcF-R126,0.2% arabinose；11 roads：VK3-FGF21-pAcF-R131,0.2% Arabinose；12 roads：VK3-FGF21-pAcF-S162,0.2% arabinose.Compiled on the position indicated by 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor Number it is according to SEQID NO：1.
Fig. 6 shows B-PER supernatant samples：1 road：VK3-FGF21 pre-induced, supernatant；2 roads：VK3-FGF21 is lured in advance Lead, bead；3 roads：Mark；4 roads：VK3-FGF210.2% arabinoses, supernatant；5 roads：VK3-FGF210.2% is Arabic Sugar, bead；6 roads：VK3-FGF21-pAcF-L10,0.2% arabinose；7 roads：VK3-FGF21-pAcF-L52,0.2% I Uncle's sugar；8 roads：VK3-FGF21-pAcF-R77,0.2% arabinose；9 roads：VK3-FGF21-pAcF-H117,0.2% is Arabic Sugar；10 roads：VK3-FGF21-pAcF-R126,0.2% arabinose；11 roads：VK3-FGF21-pAcF-R131,0.2% I Uncle's sugar；12 roads：VK3-FGF21-pAcF-S162,0.2% arabinose.It is on the Position Number indicated by 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor According to SEQ ID NO：1.
Method known to those skilled in the art can be used to purify the mutation FGF-21 albumen marked through His.Can The standard His purification of the labelled protein program provided via manufacturer come use ProBond nickel chelating resin (Invitrogen, Carlsbad, CA).
Develop pVK10 (Figure 24) to use for un-marked FGF-21 albumen, it has the sequence provided in Figure 25.This For for manufacturing the carrier of R36am and Y83am mutant and existing in instances later prominent on this non-His flag Fs GF-21 Become other data of albumen and its purifying and be shown in whole schema.
3T3-L1 is divided into fat cell and glucose uptake calibrating
To analyze the bioactivity of FGF-21 polypeptides, following calibrating can be carried out.By mouse 3T3-L1 fibroblasts (ATCC#CL-173) be seeded in the 10cm disks with the DMEM containing 10% cow's serum.Cell is set to keep not higher than 70% Density is to be expanded.Before fat cell is begun to differentiate into, cell is set to reach that 100% covers with；It must change every three days Culture medium.Cell, which is counted, and is inoculated into the density of every 25,000 cells in hole in 96 orifice plates (can also apply cell In the orifice plates of Cytostar-T 96) and cultivate again 48 hours.By added after previous culture medium is removed following culture medium come Induction differentiation：It is supplemented with 10%FBS (hyclone), 1 μM of dexamethasone (DBX), 0.5mM3- isobutyl group -1- methyl xanthines (IBMX) and 5 μ g/mL insulin DMEM.It is a kind of induce differentiation alternative be with 1 μM of Rosiglitazone processing cell and Cultivate 6 days, culture medium is then replaced by DMEM/10%FBS, be divided into because this is a kind of induction 3T3-L1 fibroblasts The faster manner of fat cell.The third possibility is to shorten divergaence time by two kinds of suites.
After the culture medium containing DBX/IBMX/ insulin is added into cell, by cell culture 48 hours.Will culture Base is replaced by DMEM/10%FBS/5 μ g/mL insulin, and by cell culture 48 hours.Then culture medium is replaced by DMEM/10%FBS and replace the culture medium with fresh culture every three days.Cell will break up 7-14 days.Noble cells Accumulate lipid drop.It can useOIL RED OBy cell dyeing.Once 95% fat cell contains lipid drop, the cell is Available for glucose uptake calibrating.
It is being supplemented with 0.1% 3T3-L1 for not handling differentiation in the BSA of fatty acids DMEM with FGF-21 (1 μ g/mL) Up to 18 hours so that cell is suffered from hunger.Then with the Kreb ' s-Ringer HEPES buffer solutions (KRH for being supplemented with 0.1%FAF-BSA =0.118M NaCl, 5mM KCl, 2.54mM CaCl2、1.19mM KH2PO4、1.19mMMgSO4With 20mM HEPES) will be thin Born of the same parents wash 3 times.By adding 4 μ Ci, 0.1mM 2- deoxidation D- [1- into KRH/0.1%FAF-BSA buffer solutions3H]-glucose To prepare mark mixture.Add cell and cultivated 1 hour at 37 DEG C.By using the ice containing 20 μM of cytochalasin Bs Cold PBS, which washes twice cell, carrys out terminating reaction.Plate is blotted to eliminate any remaining buffer.Scintillation solution is added into each hole Body and with TopCounter to sample count.
A kind of alternative for measuring glucose uptake is to load the 3T3- of differentiation with on-radiation substrate (2-NBDG) L1 cells and use fluorescence plate reader reading.A kind of indirect program for measuring glucose uptake is that measurement GLUT1 or GLUT4 exists Expression on cell membrane surface.GLUT1 and GLUT4 are that cell is transferred to from internal compartment after insulin or FGF-21 are stimulated Glucose transporter on film surface.The ELISA on liver cell can be developed.
Example 3
The introducing of amino acid of this Examples detail containing carbonyl and its subsequent reactions with the PEG containing aminooxy group.
A kind of method for producing the FGF-21 polypeptides for being associated with the non-naturally encoded amino acids containing ketone of this example displaying, it is described Polypeptide then reacts with the about 5,000MW PEG containing aminooxy group.Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7, 8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、 34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、 59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、 84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、 107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、 126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、 145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、 164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place each residue Replaced respectively by the non-naturally encoded amino acids with following structure：
Sequence for that will be incorporated to acetyl-phenylalanine locus specificity in FGF-21 is SEQ ID NO：1 And SEQ ID NO (FGF-21)：16 or 17 (muttRNA, Methanococcus jannaschii mtRNACUA Tyr), and the institute in examples detailed above 2 15,29,30 or 31 (TyrRS LW1,5 or 6) stated.
FGF-21 polypeptide variants comprising the amino acid containing carbonyl after modification with following form containing aminooxy group PEG derivatives reactions：
R-PEG(N)-O-(CH2)n-O-NH2,
Wherein R is methyl, and n is 3 and N is about 5,000MW.The purified FGF-21 to acetyl phenyl alanine will be contained 25mM MES (Sigma Chemical, St.Louis, MO) (pH 6.0), 25mM Hepes (Sigma are dissolved in 10mg/mL Chemical, St.Louis, MO) (pH 7.0) or 10mM sodium acetates (Sigma Chemical, St.Louis, MO) (pH 4.5) In, reacted with the PEG containing aminooxy group of 10 times to 100 times excess, and be then stirred at room temperature 10-16 hours (Jencks, W.J.Am.Chem, Soc.1959,81, page 475).Then, PEG-FGF-21 is diluted in appropriate buffer solution to be stood Purify and analyze.
Example 4
Engaged with the PEG by being made up of the bonded azanol base being connected with PEG of acid amides.
PEG reagents with following structure are using the program described in example 3 and the non-naturally encoded amino acids containing ketone Coupling：
R-PEG(N)-O-(CH2)2-NH-C(O)(CH2)n-O-NH2,
Wherein R is methyl, n=4 and N is about 20,000MW.Reaction, purifying and analysis condition are as described in example 3.
Example 5
Two kinds of different non-naturally encoded amino acids are introduced into FGF-21 polypeptides by this Examples detail.
The non-natural comprising ketone is incorporated at a kind of two positions produced in following residue of this example displaying to compile The method of the FGF-21 polypeptides of code amino acid：Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12, 13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、 38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、 63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、 88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、 110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、 129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、 148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、 167th, 168,169,170,171,172,173,174,175,176,177,178,179,180,181,182 (that is, in protein Carboxyl terminal) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7).Except selection codon is in nucleic acid Two different locis at introduce outside, prepare FGF-21 polypeptides as described in example 1 and example 2.
Example 6
Engagement and follow-up in-situ reducing of this Examples detail FGF-21 polypeptides with the PEG containing hydrazides.
The FGF-21 polypeptides for being associated with the amino acid containing carbonyl are the programs according to example 2 and example 3 to prepare. The PEG containing hydrazides with following structure is engaged after modification with FGF-21 polypeptides：
R-PEG(N)-O-(CH2)2-NH-C(O)(CH2)n-X-NH-NH2,
Wherein R is methyl, and n=2 and N=10,000MW, and X are carbonyl (C=O).It will contain to acetyl group phenylpropyl alcohol ammonia Acid purified FGF-21 be dissolved under the concentration between 0.1-10mg/mL 25mM MES (SigmaChemical, St.Louis, MO) (pH 6.0), 25mM Hepes (Sigma Chemical, St.Louis, MO) (pH7.0) or 10mM acetic acid In sodium (Sigma Chemical, St.Louis, MO) (pH 4.5), reacted with the PEG containing hydrazides of 1 times to 100 times excess, and And it is dissolved in H by adding2Until storing solution 1M NaCNBH of the ultimate density for 10-50mM in O3(Sigma Chemical, St.Louis, MO) the corresponding hydrazone of in-situ reducing.Reached 18-24 hours to progress reaction at room temperature at 4 DEG C in dark surrounds.Pass through The 1M Tris (Sigma Chemical, St.Louis, MO) for adding about pH 7.6 are terminated until final Tris concentration is 50mM Reaction is diluted in appropriate buffer solution to be purified immediately by reaction.
Example 7
Amino acid containing alkynes is introduced into FGF-21 polypeptides and derives it with mPEG- azide by this Examples detail Change.
Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、 44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、 69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、 94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、 114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、 133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、 152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、 171st, 172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7) place the substitution of each freedom of residue following non-naturally encoded amino acids：
Sequence for that will be incorporated to propargyl-tyrosine locus specificity in FGF-21 is SEQ ID NO：1(FGF- 21)、SEQ ID NO：16 or 17 (muttRNA, Methanococcus jannaschii mtRNACUA Tyr) and described in example above 2 22, 23 or 24.Contain the FGF-21 polypeptides of propargyl tyrosine and using the condition described in example 3 in expression in escherichia coli Purified.
Purified FGF-21 containing propargyl-tyrosine is delayed being dissolved in PB under 0.1-10mg/mL concentration 10 times to 1000 times excess are added in fliud flushing (100mM sodium phosphates, 0.15M NaCl, pH=8) and into reactant mixture PEG containing azido.Then, by the CuSO of catalytic amount4Added with copper wire in reactant mixture.Mixture through cultivate (including (but not limited to) about 4 hours at room temperature or 37 DEG C, or at 4 DEG C whole night) after, add H2O and by dialyse membrane filtration mix Compound.It may include (but not limited to) by the similar program described in example 3 to analyze the addition of sample.
In this example, PEG will have following structure：
R-PEG(N)-O-(CH2)2-NH-C(O)(CH2)n-N3,
Wherein R is methyl, and n is 4 and N is 10,000MW.
Example 8
Replace big hydrophobic amino acid in this Examples detail FGF-21 polypeptides with propargyl tyrosine.
Phe, Trp or Tyr residue present in a position in FGF-21 region below：Position 1 is (that is, at N ends End) before, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26, 27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、 52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、 77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、 102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、 121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、 140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、 159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、 178th, 179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Phase in 2-7 Answer amino acid), it is to be replaced as described in example 7 by following non-naturally encoded amino acids：
PEG is being connected to after modification on the FGF-21 polypeptide variants comprising the amino acid containing alkynes.PEG will have following Structure：
Me-PEG(N)-O-(CH2)2-N3,
And coupling procedure will comply with the program in example 7.This process can produce FGF-21 polypeptide variants, and it is included Substantially with a kind of naturally occurring isosteric non-naturally encoded amino acids of big hydrophobic amino acid and in the polypeptide Different loci at through PEG Derivatives Modifieds.
Example 9
FGF-21 polypeptides homodimers that this Examples detail is separated by one or more PEG connexons, heterogeneous two The generation of polymers, homopolymeric thing or heteromultimer thing.
Make the FGF-21 polypeptide variants and the difunctionality PEG derivatives reactions of following form containing alkynes produced in example 7：
N3-(CH2)n-C(O)-NH-(CH2)2-O-PEG(N)-O-(CH2)2-NH-C(O)-(CH2)n-N3,
Wherein n is that 4 and PEG has about 5,000 average MW, to produce two FGF-21 molecules by PEG physical separations The corresponding FGF-21 polypeptides homodimers opened.FGF-21 polypeptides can be even with one or more kinds of other polypeptides in a similar manner Close to form heterodimer, homopolymeric thing or heteromultimer thing.Will as being coupled, purified in example 7 and example 3 and Analysis.
Example 10
The coupling of this Examples detail sugar moieties and FGF-21 polypeptides.
Before position 1 (that is, in N-terminal), 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, 19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、 44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、 69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、 94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、 114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、 133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、 152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、 171st, 172,173,174,175,176,177,178,179,180,181,182 (that is, in the carboxyl terminal of protein) (SEQ ID NO：1 or in SEQ ID NO：Corresponding amino acid in 2-7) residue at place is to be compiled as described in example 3 by following non-natural Code 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor：
FGF-21 polypeptide variants comprising the amino acid containing carbonyl after modification with N- acetyl glucosamines (GlcNAc) β-connection aminooxy group analog reaction.The mixing FGF-21 in 100mM aqueous acetic acid sodium buffer solutions (pH 5.5) Polypeptide variants (10mg/mL) are sugared (21mM) with aminooxy group, and cultivated 7 hours to 26 hours at 37 DEG C.By in environment At a temperature of will engage FGF-21 polypeptides (5mg/mL) and UDP- galactolipins (16mM) and the β-Isosorbide-5-Nitrae-galactosyltransferase of sugar (0.4 units per ml) cultivates 48 hours in 150mM HEPES buffer solutions (pH 7.4) and makes second of sugar and the first carbohydrase Promote coupling (Schanbacher et al., J.Biol.Chem.1970,245,5057-5061).
Example 11
The generation of this Examples detail Pegylation FGF-21 polypeptide antagonists.
Residue (including but not limited to being combined relevant residue with FGF-21 acceptors) is by following non-as described in example 3 Natural coded amino acid substitution：
FGF-21 polypeptide variants comprising the amino acid containing carbonyl after modification with following form containing aminooxy group PEG derivatives reactions：
R-PEG(N)-O-(CH2)n-O-NH2,
Wherein R is methyl, and n is 4 and N is 20,000MW, to produce at the single site in polypeptide through PEG derivatives The FGF-21 polypeptide antagonists comprising non-naturally encoded amino acids of modification.As being coupled, being purified and being analyzed in example 3.
Example 12
It is directly connected to FGF-21 polypeptides homodimers, heterodimer, the homopolymeric thing or heterogeneous of FGF-21 molecules The generation of polymer
FGF-21 polypeptide variants comprising the amino acid containing alkynes can be with another comprising the amino acid containing azido FGF-21 polypeptide variants are directly coupled.FGF-21 polypeptides can be coupled in a similar manner with one or more kinds of other polypeptides with Form heterodimer, homopolymeric thing or heteromultimer thing.As being coupled, being purified in example 3, example 6 and example 7 And analysis.
Example 13
PEG-OH+Br-(CH2)n-C≡CR′→PEG-O-(CH2)n-C≡CR′
A B
Poly- alkane glycol (P-OH) is reacted with alkyl halide (A) and form ether (B).In these compounds, n is 1 to 9 Integer and R ' can be straight or branched, saturation or undersaturated C1 to C20 alkyl or miscellaneous alkyl.R ' or C3 to C7 saturations Or undersaturated cyclic alkyl or cycloheteroalkyl, the aryl or heteroaryl for being substituted or being unsubstituted, be substituted or without Substituted alkaryl (alkyl is C1 to C20 saturations or unsaturated alkyl) or miscellaneous alkaryl.Generally, PEG-OH be with The polyethylene glycol (PEG) or mono methoxy polyethylene glycol (mPEG) of the molecular weight of 800 dalton to 40,000 dalton (Da).
Example 14
mPEG-OH+Br-CH2-C≡CH→mPEG-O-CH2-C≡CH
With the mPEG-OH of molecular weight of NaH (12mg, the 0.5mmol) processing with 20,000Da in THF (35mL) (mPEG-OH 20kDa；2.0g, 0.1mmol, Sunbio).Then, by 80 weight % solution forms being dissolved in dimethylbenzene The KI of propargyl bromide solution (0.56mL, 5mmol, 50 equivalent, Aldrich) and catalytic amount is added in solution and is mixed gained Thing is heated to backflow and lasts 2 hours.It is subsequently added water (1mL) and removes solvent under vacuo.CH is added into residue2Cl2 (25mL) and organic layer is separated, use anhydrous Na2SO4Dry, and by reduction in bulk to about 2mL.By this CH2Cl2Solution adds dropwise Enter in ether (150mL).Gained sediment is collected, with several pieces cold ether and drying, propargyl-O-PEG is obtained.
Example 15
mPEG-OH+Br-(CH2)3-C≡CH→mPEG-O-(CH2)3-C≡CH
With the mPEG-OH of molecular weight of NaH (12mg, the 0.5mmol) processing with 20,000Da in THF (35mL) (mPEG-OH 20kDa；2.0g, 0.1mmol, Sunbio).Then, by the bromo- 1- pentynes of the 5- of 50 equivalents (0.53mL, 5mmol, Aldrich) and in the KI addition mixtures of catalytic amount.Gained mixture is heated to backflow and lasts 16 hours.It is subsequently added water (1mL) and solvent is removed under vacuo.CH is added into residue2Cl2(25mL) and organic layer is separated, with anhydrous Na2SO4Dry, and by reduction in bulk to about 2mL.By this CH2Cl2Solution is added dropwise in ether (150mL).Collect gained Sediment, with several pieces cold ether and drying, obtains corresponding alkynes.The chloro- 1- pentynes of 5- can be used in similar reaction.
Example 16
(1)m-HOCH2C6H4OH+NaOH+Br-CH2-C≡CH→m-HOCH2C6H4O-CH2-C≡CH
(2)m-HOCH2C6H4O-CH2-C≡CH+MsCl+N(Et)3→m-MsOCH2C6H4O-CH2-C≡CH
(3)m-MsOCH2C6H4O-CH2-C≡CH+LiBr→m-Br-CH2C6H4O-CH2-C≡CH
(4)mPEG-OH+m-Br-CH2C6H4O-CH2-C≡CH→mPEG-O-CH2-C6H4O-CH2-C≡CH
Powder is added in the solution being dissolved in first to 3- hydroxy-benzyl alcohols (2.4g, 20mmol) in THF (50mL) and water (2.5mL) Last shape sodium hydroxide (1.5g, 37.5mmol), and it is subsequently added the alkynes for 80 weight % solution forms being dissolved in dimethylbenzene Bromide solution (3.36mL, 30mmol).Reactant mixture is heated under reflux 6 hours.10% lemon is added into mixture Lemon acid and removes solvent at (2.5mL) under vacuo.It is with ethyl acetate (3 × 15mL) extracted residues and molten with saturation NaCl The organic layer that liquid (10mL) washing merges, uses MgSO4It is dried and concentrated, obtains 3- propargyloxy benzylalcohols.
At 0 DEG C, methane sulfonyl chloride (2.5g, 15.7mmol) and triethylamine (2.8mL, 20mmol) are added into compound 3 (2.0g, 11.0mmol) is dissolved in CH2Cl2In solution in, and reaction solution was placed into refrigerator up to 16 hours.Conventional processing Obtain the methanesulfonates in pale yellowish oil.The grease (2.4g, 9.2mmol) is dissolved in THF (20mL) and added Enter LiBr (2.0g, 23.0mmol).Reactant mixture is heated into backflow to last 1 hour and be subsequently cooled to room temperature.To mixed Water (2.5mL) is added in compound and solvent is removed under vacuo.With ethyl acetate (3 × 15mL) extracted residues and use saturation The organic layer that NaCl solution (10mL) washing merges, uses anhydrous Na2SO4It is dried and concentrated, obtains required bromide.
MPEG-OH 20kDa (1.0g, 0.05mmol, Sunbio) are dissolved in THF (20mL) and cold in ice bath The solution.NaH (6mg, 0.25mmol) is added after several minutes under vigorous stirring, the bromination obtained above is subsequently added into The KI of thing (2.55g, 11.4mmol) and catalytic amount.Remove cooling bath and gained mixture is heated to backflow to last 12 small When.Water (1.0mL) is added into mixture and solvent is removed under vacuo.CH is added into residue2Cl2(25mL) and divide From organic layer, anhydrous Na is used2SO4Dry, and by reduction in bulk to about 2mL.It is added dropwise in diethyl ether solution (150mL), produces white Color sediment, collects the sediment to obtain PEG derivatives.
Example 17
mPEG-NH2+X-C(O)-(CH2)n-C≡CR′→mPEG-NH-C(O)-(CH2)n-C≡CR′
As it appears from the above, also can be by making the polyethylene glycol polymer containing functional end-group and the reaction containing alkynes functional group Property molecule coupling obtain polyethylene glycol polymer of the end containing alkynes.N is between 1 and 10.R ' can be H or C1 to C4 small alkane Base.
Example 18
(1)HO2C-(CH2)2-C≡CH+NHS+DCC→NHSO-C(O)-(CH2)2-C≡CH
(2)mPEG-NH2+NHSO-C(O)-(CH2)2-C≡CH→mPEG-NH-C(O)-(CH2)2-C≡CH
4- pentinoic acids (2.943g, 3.0mmol) are dissolved in CH2Cl2In (25mL).Add n-hydroxysuccinimide (3.80g, 3.3mmol) and DCC (4.66g, 3.0mmol) and at room temperature by solution stirred overnight.The thick NHS esters 7 of gained are not It is used for through being further purified in next reaction.
By the mPEG-NH of the molecular weight with 5,000Da2(mPEG-NH2, 1g, Sunbio) it is dissolved in THF (50mL) simultaneously And mixture is cooled to 4 DEG C.NHS esters 7 (400mg, 0.4mmol) are added portionwise under vigorous stirring.Stir the mixture for 3 small When, while being warming up to room temperature.It is subsequently added water (2mL) and removes solvent under vacuo.CH is added into residue2Cl2(50mL) And organic layer is separated, anhydrous Na is used2SO4Dry, and by reduction in bulk to about 2mL.By this CH2Cl2Ether is added dropwise in solution In (150mL).Collect gained sediment and be dried in a vacuum.
Example 19
This example represents sulfonyl methane base ester (its methane sulfonate for being also referred to as polyethylene glycol or the first sulphur of polyethylene glycol Acid esters) preparation.Corresponding tosylate and halide can be prepared by similar program.
mPEG-OH+CH3SO2Cl+N(Et)3→mPEG-O-SO2CH3→mPEG-N3
Under a nitrogen, by mPEG-OH (MW=3,400,25g, 10mmol) azeotropic distillation 2 hours in 150mL toluene simultaneously And solution is cooled to room temperature.By the anhydrous CH of 40mL2Cl2Added with 2.1mL anhydrous triethylamines (15mmol) in solution.In ice bath It is middle to cool down the solution and methane sulfonyl chlorides (15mmol) of the 1.2mL through distillation is added dropwise.Under a nitrogen, at room temperature, By solution stirred overnight, and by adding 2mL absolute ethyl alcohols come stopped reaction.The mixture is evaporated under vacuo to remove Solvent (solvent mainly in addition to toluene), filtering is concentrated in vacuo and then precipitated in 100mL ether again.With number Part cold ether filtrate is simultaneously dried under vacuum, and obtains methanesulfonates.
Methanesulfonates (20g, 8mmol) is dissolved in 75ml THF and solution is cooled to 4 DEG C.To the solution of cooling Middle addition sodium azide (1.56g, 24mmol).Under a nitrogen, reaction is heated to backflow and lasts 2 hours.Subsequent evaporation solvent And use CH2Cl2(50mL) dilutes residue.Organic fraction is washed with NaCl solution and anhydrous MgSO is used4Dry.By volume Taper to 20ml, and product is precipitated in the absolute ether cold by adding 150ml.
Example 20
(1)N3-C6H4-CO2H→N3-C6H4CH2OH
(2)N3-C6H4CH2OH→Br-CH2-C6H4-N3
(3)mPEG-OH+Br-CH2-C6H4-N3→mPEG-O-CH2-C6H4-N3
The method described in U.S. Patent No. 5,998,595 can be used to prepare 4- azido benzylalcohols, the patent is It is incorporated herein by reference.At 0 DEG C by methane sulfonyl chloride (2.5g, 15.7mmol) and triethylamine (2.8mL, 20mmol) add 4- azidos benzylalcohol (1.75g, 11.0mmol) and be dissolved in CH2Cl2In solution in, and reaction solution is placed into Up to 16 hours in refrigerator.Conventional processing obtains the methanesulfonates in pale yellowish oil.The grease (9.2mmol) is dissolved in In THF (20mL) and add LiBr (2.0g, 23.0mmol).By reactant mixture be heated to backflow last 1 hour and with After be cooled to room temperature.Water (2.5mL) is added into mixture and solvent is removed under vacuo.Extracted with ethyl acetate (3 × 15mL) Take residue and the organic layer of merging is washed with saturation NaCl solution (10mL), use anhydrous Na2SO4It is dried and concentrated, obtains institute Need bromide.
With in THF (35mL) NaH (12mg, 0.5mmol) processing mPEG-OH 20kDa (2.0g, 0.1mmol, Sunbio the KI of bromide (3.32g, 15mmol) and catalytic amount) and into mixture is added.Gained mixture is heated to Backflow lasts 12 hours.Water (1.0mL) is added in mixture and solvent is removed under vacuo.CH is added into residue2Cl2 (25mL) and organic layer is separated, use anhydrous Na2SO4Dry, and by reduction in bulk to about 2mL.Diethyl ether solution is added dropwise In (150mL), sediment is produced, collects the sediment to obtain mPEG-O-CH2-C6H4-N3。
Example 21
NH2-PEG-O-CH2CH2CO2H+N3-CH2CH2CO2-NHS→N3-CH2CH2-C(O)NH-PEG-O-CH2CH2CO2H
By NH2-PEG-O-CH2CH2CO2H (MW 3,400Da, 2.0g) is dissolved in NaHCO3In saturated aqueous solution (10mL) simultaneously And solution is cooled to 0 DEG C.Propionic acid 3- azido -1-N- hydroxysuccinimide eaters (5 equivalent) are added under vigorous stirring.3 After hour, 20mL H are added2O and stir the mixture for again at room temperature 45 minutes.Use 0.5N H2SO4By pH value regulation to 3 And NaCl is added until concentration is about 15 weight %.Use CH2Cl2(100mL × 3) extractive reaction mixture, uses Na2SO4Dry And concentrate.After being precipitated with cold diethyl ether, product is collected by filtration and is dried under vacuum, obtain ω-carboxyl-azide PEG Derivative.
Example 22
mPEG-OMs+HC≡CLi→mPEG-O-CH2-CH2-C≡C-H
Under vigorous stirring, to prepared by as known in art and being cooled to -78 DEG C of acetylene lithium (4 equivalent) and be dissolved in The mPEG-OMs solution being dissolved in THF is added dropwise in solution in THF.After 3 hours, reaction is set to be warming up to room temperature and pass through Add 1mL butanol stopped reactions.It is subsequently added 20mL H2O and stir the mixture for again at room temperature 45 minutes.Use 0.5N H2SO4By pH value regulation is to 3 and adds NaCl until concentration is about 15 weight %.Use CH2Cl2(100mL × 3) extractive reaction Mixture, uses Na2SO4It is dried and concentrated.After being precipitated with cold diethyl ether, product is collected by filtration and is dried under vacuum, 1- is obtained (butyl- 3- alkynyls epoxide)-methoxy poly (ethylene glycol) (mPEG).
Example 23
The method described in documents below can be used to select the amino acid containing azido and the amino acid sites containing acetylene It is incorporated in protein to property：L.Wang et al., (2001),Science292：498-500；J.W.Chin et al.,Science301：964-7(2003)；J.W.Chin et al., (2002),Journal of the American Chemical Society124：9026-9027；J.W.Chin and P.G.Schultz, (2002),Chem Bio Chem11：1135-1137； J.W.Chin et al., (2002),PNAS United States of America99：11020-11024；And L.Wang and P.G.Schultz, (2002),Chem.Comm.1：1-11.Being incorporated to after amino acid, immediately at 37 DEG C, spreading out there is 2mM PEG Biological, 1mM CuSO4In the case of about 1mg copper wires, entered with the 0.01mM protein in phosphate buffer (PB) (pH 8) Row cycloaddition reaction was up to 4 hours.
Example 24
This example is described to acetyl group-D, the synthesis of L-phenylalanine (pAF) and m-PEG- hydroxylamine derivatives.
Using being previously described in Zhang, Z., Smith, B.A.C, Wang, L, Brock, A., Cho, C. and Schultz, P.G., Biochemistry, (2003) 42, program in 6735-6746 synthesizes racemic pAF.
For synthesis m-PEG- hydroxylamine derivatives, following procedure is completed.To (the N-t- that 1 hour is stirred under room temperature (RT) Boc- aminooxy groups) acetic acid (0.382g, 2.0mmol) and 1,3- Diisopropylcarbodiimide (0.16mL, 1.0mmol) be dissolved in two Added in solution in chloromethanes (DCM, 70mL) methoxypolyethylene glycol amine (m-PEG-NFb, 7.5g, 0.25mmol, Mt.30K, from BioVectra) and diisopropylethylamine (0.1mL, 0.5mmol).Reactant is stirred at room temperature 48 small When, and then it is concentrated to about 100mL.Mixture is added dropwise in cold diethyl ether (800mL).Sunk through the t-Boc products protected Form sediment out and be collected by filtration, washed with ether (3 × 100mL).By being dissolved in again in DCM (100mL) and in second It is further purified twice in precipitation in ether (800mL).Product is dried in a vacuum, obtains 7.2g (96%), passes through NMR and indenes Triketone experiment (Nihydrin test) is confirmed.
The removings of the Boc through protecting product (7.0g) obtained above are to be carried out at 0 DEG C in 50%TFA/DCM (40mL) 1 hour and then at room temperature carry out 1.5 hours.Remove in a vacuum after major part TFA, by adding two into residue 4N HCl in oxane (1mL) and the tfa salt of hydroxylamine derivative is converted into hydrochloride.Sediment is dissolved in DCM (50mL) In and precipitate again in the ether (800mL).Final product (6.8g, 97%) is collected by filtration, with ether (3 × 100mL) wash, be dried in a vacuum, store under a nitrogen.Other PEG (5K, 20K) azanols are synthesized using identical program to derive Thing.
Example 25
The analysis of the ERK1/2 phosphorylations induced by FGF-21WT and 30K PEG analogs：
With the density (DMEM+10%FBS) of every 100,000 cells in hole by through mankind's Klotho beta stable transfections 293 cells are inoculated into poly-Lys coated panels.Second day, cell reached that 100% covers with, and suctioned out culture medium and with fresh training Support base to replace, and cultivate whole night.After 24 hours, stimulate thin with appropriate 30K PEGFGF-21 analogs using standard FGF21WT Born of the same parents.Each individual compound is prepared by being diluted in PBS/1%BSA.At 37 DEG C, in insulating box, in triplicate will Cell is handled 10 minutes.After cultivating 10 minutes, culture medium is carefully suctioned out from each hole, and 40 μ l are added into each hole and is contained Cold 1 × cell signalling dissolving buffer solution (PI mixed liquors, Na3VN4 and PMSF) of protease/inhibitors of phosphatases is with production Raw cell lysis liquid.By 96 orifice plates be placed on ice up to 20 minutes and then under 4000rpm rotate 10 minutes.At -80 DEG C Lower frozen cell lysate.Each sample is thawed and is added 10 μ l cell lysis liquid later and scribble capture unphosphorylated form ERK1/2 and phosphorylation form ERK1/2 antibody the plate handled through MSD in.With Primary antibodies cultivate 2 hours, then Plate is washed for several times with specific buffer solution, secondary antibody is then added.After cultivating 1 hour, then by plate washing for several times.It will be used to read Several buffer solutions is added in each hole.Plate is transferred in MSD reading machines.Produced curve is read based on anti-phosphorylated CREB 1/2 Take unit and calculate EC50 using Sigma Plot.By using WT EC50 except 30K Pegylation specific compounds EC50 calculates loss of activity multiple.
Example 26：The phosphorylations of cell ERK 1/2 examine and determine (pERK) scheme and MSD analyses
293 β Klotho-4 cells are maintained at DMEM+10%FBS+P/S+0.5mg/mL Geneticins (Geneticin) In.When cell reaches that 50-90% is covered with, make its trypsinized, and use is inoculated into the density of every 100,000 cell in hole In DMEM+10%FBS+P/S in 96 orifice plates of poly-D-lys coatings.
Second day, when cell about 100% is covered with, it is carried out to check to ensure that culture medium is still red, then suctioned out Culture medium, the serial dilution (being diluted in PBS+1%BSA) of every μ L of hole 200 FGF-21 variants is moved into 96 orifice plates. 96 orifice plates are then placed into 37 DEG C, 5%CO2In insulating box, 9 minutes are accurately lasted.FGF-21 processed materials are then suctioned out completely And freshly prepared 1 × cell signalling dissolving buffer solution+1 × Sigma protease inhibitors per the μ L of hole 40 is added to mix Close liquid+2mM sodium orthovanadates+1mM PMSF+1 × MSD inhibitors of phosphatases I+1 × MSD inhibitors of phosphatases II+1 × MSD albumen Enzyme inhibitor mixed liquor+2mM MSD PMSF.Disk is placed on ice, and shelved 25 minutes, while with P20 pipettes to each hole Aspirate to mix lysate above and below progress.Make after each hole mixing, whole ice bucket and disk are placed into 4 DEG C of vibrations within remaining time On device.After 25 minutes, plate is set to be rotated 10 minutes under 4000rpm at 4 DEG C.Supernatant is transferred to the round bottom 96 of cooled on ice In orifice plate.
MSD is analyzed
Use the full cell lysis liquid kit Phospho of Meso Scale Discovery MULTI-SPOT verification systems (T/Y 202/204；185/187)/Total-ERK1/2/ calibratings are examined and determine to carry out this.
Make at room temperature all MSD reagents thaw and prepared according to kit specification it is all necessary to buffer solution. In each hole that 150 μ L blocking agents A are added to phosphoERK-totalERK duplex boards, and make it at room temperature in oscillator It is upper to block 1 hour.Then, plate is washed 4 times with every μ L of hole 160 1 × lavation buffer solution.The dissolving per the μ L of hole 16 is added to buffer Liquid+protease and inhibitors of phosphatases (preparing to carry out cytositimulation in early time), and by every μ L of hole 10 from cold bacteriolyze It is transferred in liquid supernatant plate in the hole of MSD plates (cumulative volume subsequently becomes every μ L of hole 26).By MSD plates at room temperature in oscillator It is upper to cultivate 3 hours, then plate is washed 4 times with 1 × lavation buffer solution per the μ L of hole 160.Add the detection antibody per the μ L of hole 25 (diluting 50 times with antibody dilution buffer) and setting are cultivated 1 hour on the oscillator at room temperature.Use again per the μ L of hole 160 Plate is washed 4 times and adds 1 × reading buffer solution T per the μ L of hole 150 by 1 × lavation buffer solution, immediately uses MSD Sector The machines of Imager 2400 are to plate reading.
BCA is quantified
Use Pierce BCA protein assay kits.Buffer solution is dissolved using 1 × cell signalling, it is dilute with twice Along post downwards, since 2mg/mL top concentration, (last group of hole is slow to dilution BSA reference materials to the mode released in 96 orifice plates Fliud flushing, no BSA).Every μ L of hole 25 BSA reference materials are moved in two MaxiSorp96 orifice plates in duplicate.With 1 × thin Born of the same parents' signal transduction dissolving buffer solution is carried out 3 times of dilutions of lysate and added with every μ L of hole 25 in MaxiSorp plates.According to Pierce kit instruction cards carry out preparation work reagent, and 200 μ L are moved in each hole of MaxiSorp plates.Cultivate at room temperature The plate and with plate reader under λ=562nm reading.
Data analysis
The concentration of all lysates is calculated with BCA kits.If the concentration of lysate is all similar, then it is not necessary on MSD analyses are corrected.For MSD analyses, copy-point is averaged, standard deviation and CV values is calculated.Use SigmaPlot Come the EC50 values of the serial dilution that calculates FGF-21 variants, and multiple higher than WT EC50 is used as variant Scaling Standards.As a result it is found in Fig. 7 a and Fig. 7 b of present application.
Example 27：Un-marked FGF-21 downstream process
Inclusion body prepares dissolving
Cell paste is set to be re-suspended into 4 DEG C of inclusion body (IB) buffer solution I (50mM Tris, pH 8.0 by mixing； 100mM NaCl；1mM EDTA；1%Triton X-100；4 DEG C) in, reach final 10% solid concentration.By making to hang again Float matter amounts to twice to dissolve cell through Micro Fluid bed (micro fluidizer), then centrifugation (10,000g；15 points Clock；4 DEG C) and it is decanted off supernatant.By making IB beads be re-suspended into IB buffer solutions I (the 50mM Tris, pH of another volume 8.0；100mM NaCl；1mM EDTA；1%Triton X-100；4 DEG C) in wash IB beads, and wear settling flux material Cross Micro Fluid bed to amount to twice, then centrifugation (10,000g；15 minutes；4 DEG C) and it is decanted off supernatant.Make IB bead settling flux To buffer solution II (the 50mM Tris, pH 8.0 of 1 volume；100mM NaCl；1mM EDTA；4 DEG C) in, then centrifuge (10, 000g；15 minutes；4 DEG C) and it is decanted off supernatant.Then, IB beads are made to be re-suspended into the buffer solution II (50mM of 1/2 volume Tris, pH 8.0；100mMNaCl；1mM EDTA；4 DEG C) in.IB is distributed in appropriate containers, (10,000g are then centrifuged； 15 minutes；4 DEG C) and it is decanted off supernatant.It (now can otherwise be stored at -80 DEG C until entering by solubilization of inclusion bodies One step is used).
Solubilization of inclusion bodies
With dissolving buffer solution (20mM Tris, pH 8.0；8M urea；10mM β-ME) by solubilization of inclusion bodies to ultimate density For 10-15mg/mL, and the IB of dissolving is cultivated 1 hour under constant mixing at room temperature.By filtering (0.45 μm of PES Filter) remove insoluble substance and carry out regulatory protein (when protein concentration is high) by using other dissolving buffer solution dilutions Matter concentration (and not always required).
Refolding
By using 20mM Tris (pH 8.0；4 DEG C) final protein concentration is diluted to for 0.5mg/mL to be rolled over again It is folded.Make its refolding 18 hour to 24 hours at 4 DEG C.
Purifying
Refolding reaction solution is filtered by 0.45 μM of PES filter.It is real to buffer A (20mM Tris, pH 7.5) Q HP posts (GE Healthcare) the load material now balanced.With 20CV to 100% buffer B (20mM Tris, pH 7.5； 250mM NaCl) linear gradient elution FGF-21.Collect monomer FGF-21.
Pegylation and purifying
Take Q HP ponds and by buffer-exchanged be 20mM Tris (pH 8.0)；2M urea；1mM EDTA.Use 50% ice PH value is dripped to 4.0 by acetic acid.By sample concentration to 4.0 ± 1.0mg/mL.The PEG and most of 12: 1 molar excess is added into sample Final concentration of 1% acethydrazide (pH 4.0).Cultivated 48-72 hours at 28 DEG C.Add final 50mM's into PEG reaction solutions Tris alkali, and dilute 10 times with reverse osmosis water (RO water).Ensure conductivity ＜ 1mS/cm and pH value between 8.0-9.0 it Between.Source 30Q posts (GE Healthcare) loaded article of balance is realized to buffer A (20mM Tris, pH 8.0) Matter.With 20CV to 100%B (20mM Tris, pH 8.0；100mM NaCl) linear gradient elution PEG-FGF-21.Collect PEG-FGF-21 and by buffer-exchanged be 20mM Tris (pH 7.4)；150mM NaCl.PEG materials are concentrated to 1- 0.22 μm of PES filter of 2mg/mL and use carries out filter sterilization.It is stored at 4 DEG C.For extension storage, snap frozen is simultaneously It is stored at -80 DEG C.
Example 28
The pharmacokinetic property of FGF-21 compounds in rats
There is provided using this scheme on natural FGF-21 compounds and by Ambrx proprietary technologies (Ambrx ' Sproprietary technology) the FGF-21 compounds modified through PEG that produce move in the medicine that conduit is inserted in rat The data (seeing in Figure 11-23) of mechanical property.Pass through the mankind to carrying out the blood serum sample that particular point in time is obtained after self administration of medication There is FGF-21 specific ELISA to carry out the pharmacokinetics of verification test thing.
Tester：
1.Ambrx compounds PEG-R77FGF-21 will be used with the 0.25mg/ml diluted in 1 × PBS concentration.
2.Ambrx compounds PEG-Y104FGF-21 will be used with the 0.25mg/ml diluted in 1 × PBS concentration.
3.Ambrx compounds PEG-R126FGF-21 will be used with the 0.25mg/ml diluted in 1 × PBS concentration.
Tester quality/composite：
Stock concentrations=
1.0mg/mL PEG-R77FGF-21
1.16mg/mL PEG-Y104FGF-21
1.08mg/mL PEG-R126FGF-21
Animal：
12 before the study began body weight be about 250-275 grams male Sprague-Dawley (SD) rats reach There should be the jugular vein conduit by surgical installation before Ambrx.Animal in shape is to obtain and grinding from CRL Study carefully and research place is adapted it to before beginning at least 3 days.On the day of tester administration, to rat weight.Animal is housed in standard Pathogen-free domestic environment in, it is arbitrarily taken food food and water.
Animal groups：All compounds are all by administration under longitude.
1st group (n=4)：PEG-R77, is subcutaneously injected (0.25mg/kg).
2nd group (n=4)：PEG-Y104, is subcutaneously injected (0.25mg/kg).
3rd group (n=4)：PEG-R126, is subcutaneously injected (0.25mg/kg).
Animal is weighed before administration tester.Compound is allocated so as to by 1X BW (in terms of μ L) administration.To subcutaneously it throw With tester be expelled to omoplate the back of the body region in.Animal is by the single injection (time=0) of acceptance test thing.In particular point in time (seeing below), takes whole blood from animal, collects in SST Microtainer collection tubes.Make serum condensation 30 minutes, then Centrifuged.By serum transfers into polypropylene buret, with microstrip sealing, and it is stored at -80 DEG C and is carried out until with ELISA Analyze to determine FGF-21 serum-concentrations.
Data acquisition/terminal：
Each animal is all by for complete PK time-histories.About 0.25mL whole blood is taken from jugular vein conduit.In blood collection Afterwards soon, with 0.1mL normal saline flushing conduits.According to the expected drug kinetic profile of tester, acceptance test thing material Animal need following acquisition time：
Before blood sampling, 1 after administration, 2,4,8,24,32,48,56,72 and 96 hours.
Terminate：
, will be to all euthanizing animals after the completion of research.
As a result：
As a result it is provided in the schema that following table and present application are enclosed.
FGF21PEG isomers PK properties (intravenous injection)
FGF21PEG isomers PK properties (hypodermic injection)
The Tmax increases of PEGylated compounds.
The T1/2 increases of PEGylated compounds.
The AUC increases of PEGylated compounds.
Pegylation sites dependence PK attributes.
Example 29
Pegylation FGF-21 in vivo research
By PEG-FGF-21, unmodified FGF-21 and cushioning liquid administration mouse or rat.As a result will display, this hair Bright Pegylation FGF-21 has excellent activity and the half-life period extended compared with unmodified FGF-21.It is similar Ground, by the FGF-21 through modification, unmodified FGF-21 and cushioning liquid administration mouse or rat.
Pharmacokinetic analysis
The pharmacokinetic study that the descriptions of WO 2005/091944 can be carried out with the FGF-21 compounds of the present invention.By quiet In arteries and veins or FGF-21 polypeptides from subcutaneous route to the mouse administration present invention.Multiple time points before administration and upon administration To animal blood taking.Gather the blood plasma of each sample and analyzed by radioimmunoassay.Can calculate elimination half-life period and The various forms of FGF-21 polypeptides comprising non-naturally encoded amino acids and wild type FGF-21 or of the present invention FGF-21 polypeptides it Between be compared.Similarly, can be to machin administration FGF-21 polypeptides of the invention.Before administration and upon administration many Individual time point is to animal blood taking.Gather the blood plasma of each sample and analyzed by radioimmunoassay.
The polypeptide administration of the present invention to ZDF male rats (can be suffered from the obese rat of diabetes；When studying beginning 8 weeks Age, Charles River-GMI).Rat is set arbitrarily to take food the feeds of Purina 5008.Set up following test group：Physiological saline； Insulin, daily 4U；FGF-21, daily 500 μ g, it is acute (acute administration group be administered once and upon administration T=0,2,4, Take a blood sample within 8 and 24 hours)；FGF-21, daily 100 μ g；FGF-21, daily 250 μ g；FGF-21, daily 500 μ g；FGF-21 is (daily Once), 500 μ g/ml；Few fat physiological saline；Few fat insulin, daily 4U；Few fat FGF-21, daily 500 μ g.Few fat Fat group represents non-diabetic, few fat, ZDF rats.
Compound is subcutaneously injected twice daily, wherein group of the exception for second daily 500 μ g, it is in research duration (7 days) receive a shot daily.To control rats injection mediator (PBS；0.1ml).After administration 7 days, animal is set to be subjected to mouth Take glucose tolerance test.The glucose and glycerine three in blood are gathered clamping blood sampling by tail in the case of not anaesthetizing Ester.FGF-21 polypeptides can reduce plasma glucose levels with dosage-dependent manner.In addition, the rat phase with administration insulin Than few fat ZDF rats may not become hypoglycemia after the FGF-21 polypeptides of the contact present invention.
Ob/ob obesity models
Ob/ob mouse models are the animal models of hyperglycemia, insulin resistance and obesity.Can be in ob/ob mouse Measure the plasma glucose levels after being handled with FGF-21 polypeptides (compared with mediator and insulin control group).In this obesity mould In type, (0.1ml, twice daily) individually mediator was subcutaneously injected to male ob/ob mouse (7 week old) test group in 7 days (PBS), insulin (daily 4U) or FGF-21 polypeptides (daily 5 μ g and daily 25 μ g).At the 1st day, the 3rd day and the 7th day, One compound injection one hour after clamps blood sampling by tail and gathers blood, and measures using standard scheme plasma glucose syrup It is flat.If the FGF-21 polypeptides of the present invention reduce plasma glucose levels compared with mediator control group, then it stimulates glucose Intake.After with the processing of the FGF-21 polypeptides of the present invention, it may compare triglyceride levels (compared with other molecules).Can be by many Dosage, continuous infusion or single dose etc. are planted to mouse administration polypeptide.
Example 30
The pharmacokinetic evaluation of FGF21 analogs：Assess in rats with different PEG bond sites The pharmacokinetic property of 30KPEG-pAF (N6-His) FGF21 analogs.The other parameters studied are PEG MW, Yi Jisuo The dosage of administration compound.Determine the biological usability percentage of some 30KPEG-pAF (N6-His) FGF21 variants.
Animal：According to management of laboratory animal and the use committee (Institutional Animal Care and UseCommittee the scheme) ratified carries out all zooperies.Male (175-300g) Sprague-Dawley rats are Obtained from Charles River Laboratories.Rat is housed in individually to the interior with 12 hours illumination/dark cycles Cage in, and adapt it to before experiment Ambrx raising farms at least 3 days.Animal is set arbitrarily to take food qualified Purina rodents 5001 and water.
Administration and serum collection：Before transportation, conduit is installed to by CRL by performing the operation in jugular vein to carry out blood Collection.After conduit is successfully opened, animal is assigned to each treatment group before administration.With 1mL/kg dose volume through vein Compound interior or through subcutaneous administration single dose.Use storage specified in grant a certificate (Certificate of Release) Standby liquid concentration, compound dose concentration is obtained by using PBS dilutions.In Each point in time, blood is gathered by the conduit of presence Sample and it is placed into SST micro-centrifuge tubes.In collected after centrifugation serum, and it is stored at -80 DEG C until analysis.
Pharmacokinetic analysis：Ambrx Inc., La Jolla, CA are developed in Sprague-Dawley rat blood serums Quantitative PEG-FGF-21 calibrating.Use Goat anti human's class FGF-21IgG polyclonal antibodies (PAb as capture agent；RnD Systems, be sheerly AF2539) it is coated with the hole of microplate.(all it is by inciting somebody to action by reference material (STD) and quality control (QC) sample PEG-FGF-21 analogs are added in 100%Sprague Dawley rat blood serums to prepare) and study sample I- Block buffer solutions 1: 100 are loaded into hole after pre-processing.The FGF- in STD, QC and study sample is captured by fixed PAb 21.Uncombined material is removed by washing each hole.By biotin Goat anti human class FGF-21IgG PAb (RnD Systems, It is sheerly BAF2539) add in hole, washing step is then carried out, and add streptavidin horseradish peroxidase (SA-HRP；RnD Systems, catalog number (Cat.No.) DY998) to detect the PEG-FGF-21 of capture.After another washing step, Tetramethyl benzidine (TMB, Kirkegaard PerryLaboratories) substrate solution is added in hole.TMB exists in HRP It is lower with peroxide reactions and produce and the amount of PEG-FGF-21 analogs that is combined in the initial step by capture agent into The colorimetric signal of direct ratio.By adding 2N sulfuric acid come color development stopping and at 450 nm measurement colouring intensity (optical density, OD). It is being regulated and controled by computer software with standard curve in same plate comparison come realization sample and QC OD units to concentration Conversion, returned using SOFTmax Pro v5 data reductions external members according to 5 parameter logistic regression models.With ng/mL Concentration unit reported result.
Also can be examined and determine by dual anti-interlayer body or those skilled in the art known to other methods measure concentration.Make Concentration is calculated with the standard curve produced by the compound of corresponding administration.Using modeling program WinNonlin (Pharsight, 4.1 editions) assess pharmacokinetic parameter.Individual animal data are carried out using using linear (on)/logarithm (under) trapezoidal integrate Non- compartment model analysis (Noncompartmental analysis), and Weighted Uniform concentration data.
Conclusion：WT N6-His FGF21 pharmacokinetic property, which meets in Kharitonenkov et al., 2005, to be reported The property led and suitable with un-marked WT FGF21 albumen.
Compared with the result obtained by WT (without Pegylation) FGF21, made by adding 30kDa PEG molecules The pharmacokinetic profile of 30KPEG-pAF (N6-His) FGF21 isomers is dramatically increased.
When with 0.25mg/kg dosage through subcutaneous administration, PEGylated compounds show dramatically different PK overviews. H87 and L86 tend to have the PK attribute poorer than other isomers.In addition, R131 and Q108PEG30 compounds are differentially produced Raw excellent PK properties.More particularly, these compounds have AUC, Cmax and terminal half-life of improvement.Go deep into structure- Activity analysis can disclose the structure explanation of the various PK properties of Isomers.
The comparison of PEG molecular weight shows that 30KPEG-pAF91 (N6-His) FGF21 has than 20KPEG-pAF91 (N6- His) circulation persistence bigger FGF21.However, because 20kDa isomers has higher cmax value, therefore both compounds Total AUCinf can be suitable.The biological usability of 20kDa hypotypes is slightly better than 30kDa variant, and respectively 30% and 20%.
Table 3. is with the Pharmacokinetic parameter values of compound of the 0.25mg/kg dosage through subcutaneous administration rat
The curve of serum-concentration and time is assessed by non-compartment model analysis (Pharsight, 4.1 editions).Every kind ofization N=3-4 rat of compound.ND：Do not carry out；NE：Do not assess.Tmax：Reach Cmax time；Cmax：Cmax；Latter stage t1/2：Terminal half-life；AUCFinally：Area under the concentration time curve at final plasma sample/time point；AUCinf：It is extrapolated to Area under infinitely great concentration time curve；MRT：Mean residence time；Cl/f：Apparent total plasma clearance；Vz/f：End Apparent volume of distribution in stage phase.
Table 4. is with 20KPEG-pAF91 (N6-hHis) FGF21 of 0.25mg/kg dosage through subcutaneous administration rat medicine Dynamic parameter value
The curve of concentration and time is assessed by non-compartment model analysis (Pharsight, 4.1 editions).ND：Do not carry out； NE：It can not assess.Tmax：Reach Cmax time；Cmax：Cmax；Latter stage t1/2：Terminal half-life；AUCFinally：Final blood Area under the concentration time curve at slurry samples/time point；AUCinf：It is extrapolated to the face under the concentration time curve of infinity Product；MRT：Mean residence time；Cl/f：Apparent total plasma clearance；Vz/f：Apparent volume of distribution in reaching advanced stages.
Example 31
Human clinical's examination of the security and/or effect of Pegylation FGF-21 comprising non-naturally encoded amino acids Test.
PurposeIn order to observe the pegylated recombinant human FGF- comprising non-naturally encoded amino acids through subcutaneous administration 21 security and pharmacokinetics.
Patient18 health that the age is between 20-40 Sui and body weight is between 60-90kg are recruited under study for action Volunteer.The individual would not urinate toxicology with clinically notable exception for hematology or serum chemistry and feminine gender The experiment value of screening, HIV screenings and HbsAg.It should not have any following sign：Hypertension；It is any primary Property hematologic disease history；Severe liver disease, nephrosis, angiocardiopathy, gastrointestinal disease, disease of the genitourinary system, metabolic disease, nerve Medical history；Anaemia or epilepsy medical history；There is known mistake to the product, PEG or human serum albumin of bacterium or mammal source Quick property；Habituation and Heavy User to the beverage containing caffeine；Any other clinic is participated in enter research 30 days Test or through transfusing blood or donating blood；FGF-21 is once contacted in enter research 3 months；It is ill in enter research 7 days；And Physical examination or have in enter research 14 days before research during clinical laboratory assessments significantly abnormal.Can be to all individuals Security is assessed, and timing acquiring is used for the whole blood collection thing of pharmacokinetic analysis.All researchs are all in mechanism Ethics Committee ratifies and patient agrees to lower carry out.
Research and designIt should be stage i in healthy male volunteers, single center, open-label, randomness, two The crossing research in individual cycle.18 individuals are randomly assigned to (every group of 9 individuals) in one group in two treatment sequence groups.Make With the Pegylation FGF-21 comprising non-naturally encoded amino acids of equal dose and selected commercially available prod, in upper leg portion warp Two separated administration phases are with subcutaneous bolus form administration FGF-21.The dosage and frequency of administration commercially available prod are according to packaging mark Carried out indicated by label.Can by added under study for action including other group of individuals other dosage using commercially available prod, to Medicine frequency or required other parameters.Each administration phase was separated by the balance period of 14 days.For each period of two administration phases For, individual was limited in research center in 72 hours after at least 12 hours and administration before administration, but between administration phase Need not be such.If there is also other dosage, frequency or other parameters, then can also add other group of individuals to test poly- second two Refine FGF-21.FGF-21 experiment deployment thing is the Pegylation FGF-21 comprising non-naturally encoded amino acids.
Blood samplingBefore and after administration FGF-21, pass through direct venipuncture continuous blood sampling.Before administration about 30 minutes, 20 minutes and 10 minutes (3 baseline samples) and upon administration substantially following time：30 minutes and 1 hour, it is 2 small When, 5 hours, 8 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours and 72 small When, obtain the venous samples (5mL) for determining serum FGF-21 concentration.Each blood serum sample is divided into two aliquots.Will All blood serum samples are stored at -20 DEG C.Blood serum sample is transported with dry ice.The 1st day initial administration not long ago, the 4th day morning, Administration in 16th day was not long ago and the 19th day morning carried out empty stomach clinical trial test (hematology, serum chemistry and urinalysis).
Bioanalytical methodSerum FGF-21 concentration is determined using ELISA kit.
Security testEvery time administration (the 1st day and the 16th day) before and every time be administered after 6 hours, 24 hours, 48 hours and 72 hours instant recording vital signs.Security test is incidence and type and clinic based on adverse events Change of the experiment test compared with baseline.In addition, assess vital sign measurement (including blood pressure) and Physical examination results and The change compared before research.
Data analysisDuring by being subtracted from each value after administration by being administered first 30 minutes, 20 minutes and 10 minutes The FGF-21 levels average baselining FGF-21 concentration averaging and determine of three samples of collection and on baseline before administration FGF-21 concentration come correct administration after serum concentration.If serum FGF-21 concentration is less than the quantitative level examined and determine before administration, So it is not included in the calculating of average value.Medicine is determined by the serum concentration data corrected on baseline FGF-21 concentration Kinetic parameter.Using the latest edition of BIOAVL softwares, by using Digital EquipmentCorporation VAX The methods independent of model of 8600 computer systems calculates pharmacokinetic parameter.Determine following pharmacokinetics ginseng Number：Peak serum concentration (Cmax)；There is the time (t of peak serum concentrationmax)；By using linear trapezoidal rule calculate from To the last blood sampling time (AUC during time zero0-72) concentration time curve (AUC) under area；And it is fast according to eliminating The latter stage of rate constant calculations eliminates half-life period (t1/2).In latter stage in the range of linearity of Log-Linear concentration time curve, pass through Elimination rate constant is estimated in the linear regression at consecutive numbers strong point.For treating each time, the flat of pharmacokinetic parameter is calculated Equal standard deviation (SD) and the coefficient of variation (CV).Ratio (the saved composite/not saved allotment of calculating parameter average value Thing).
Safety resultsIn each treatment group, the incidence distribution of adverse events is identical.It is real with clinic before baseline or research Test examination or blood pressure are compared without clinically significant change, and Physical examination results and vital signs measurement result are compared with before research Also without significant changes.The security overview of Liang Ge treatment groups apparently should be similar.
Pharmacokinetics resultsAt each time point, measure all 18 individuals and receiving to include non-naturally encoded amino acids Pegylation FGF-21 after average serum FGF-21 concentration time curves (do not repaiied on baseline FGF-21 levels Just).All individuals all should be with baseline FGF-21 concentration before the administration in the range of normal physiologic.According on being put down before administration Sera data that equal baseline FGF-21 concentration is corrected determines pharmacokinetic parameter, and determines CmaxAnd tmax.Selected The average t of any clinical comparativemaxAll than the t of the Pegylation FGF-21 comprising non-naturally encoded amino acidsmaxSignificantly more It is short.Compared with the terminal half-life of the Pegylation FGF-21 comprising non-naturally encoded amino acids, the preclinical ratio tested Value compared with the terminal half-life of thing is significantly shorter.
Although the research of the present invention is carried out in healthy male individual, it is anticipated that class will occur in other PATIENT POPULATIONs As Absorption Characteristics and security overview；Sex patient, youngster of the colony for example with cancer or chronic renal failure Virgin patients with renal failure, the patient in autologous stored Procedure or plan carry out the patient of elective surgery.
In a word, the Pegylation FGF-21 comprising non-naturally encoded amino acids of the single dose through subcutaneous administration should be Safety, and it is well tolerable by healthy male individual.It is special according to the relative incidence, clinical trial value, life of adverse events Seek peace Physical examination results, the FGF-21 of the commercial form and Pegylation FGF-21 comprising non-naturally encoded amino acids peace Full property overview should be suitable.Pegylation FGF-21 comprising non-naturally encoded amino acids is latent to patient and healthcare provider Huge clinical efficacy is provided on ground.
It will be appreciated that example specifically described herein and embodiment are merely for illustrative purpose and will be prompted to skill to art The various modification and transformations and the modification and transformation that art personnel make according to it be included in present application spirit and authority and In the range of additional claims.Cited all publication in present application, patent, patent application case and/or other Document is incorporated herein in entirety by reference for all purposes, and the degree of reference is just as individually by each disclosure Case, Patent Case, patent application case and/or other documents are herein incorporated by reference typically for all purposes.
Table 5：Cited sequence
SEQ ID# Sequence names
1 The amino acid sequence (p-shaped formula) of FGF-21 without targeting sequencing HPIPDSSPLLOFGGOVRORYLYTDDAOOTEAHLEIREDGTVGGAADOSPE SLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLE DGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGI LAPQPPDVGSSDPLSMVGPSQGRSPSYAS
2 The amino acid sequence (p-shaped formula) of FGF-21 without targeting sequencing-marked through His MHHHHHHSGGHPIPDS SPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDG TVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFD PEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLP GLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
3 The amino acid sequence (p-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 3 leucines (the p-shaped formula of 209 amino acid) MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYL YTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTS RFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGN KSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQ GRSPSYAS
4 The amino acid sequence (p-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 2 leucines MDSDETGFEHSGLWVSVLAGLLGACQAHPIPDSSPLLQFGGQVRQRYLY TDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSR FLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNK SPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQG RSPSYAS
5 The amino acid sequence (L-shaped formula) of FGF-21 without targeting sequencing His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr GIu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
6 The amino acid sequence (L-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 3 leucines (the L-shaped formula of 209 amino acid) Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
7 The amino acid sequence (L-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 2 leucines (the L-shaped formula of 208 amino acid)
Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser Val Leu Ala Gly Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
8 The nucleotide sequence (p-shaped formula) of FGF-21 without targeting sequencing CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTC CGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCA CCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGA GCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTC AAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATG GGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCC GGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCC ACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGAC CCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCC CCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGAT GTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGA AGCCCCAGCTACGCTTCCTGA
9 The nucleotide sequence (p-shaped formula) of FGF-21 without targeting sequencing-marked through His ATGCATCATCATCATCATCATAGCGGCGGCCACCCCATCCCTGACTCC AGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTAC ACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGA TGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGC AGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAG ACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCG CTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAG GACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCAC CTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACC AGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCC ACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCC TCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTC CTGA
10 The nucleotide sequence (p-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 3 leucines ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCT GTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCT GACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTAC CTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAG GGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTC TCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAG TCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATG GATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTC TTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGC TGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGA GGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCG GAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCG GACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTAC GCTTCCTGA
11 The nucleotide sequence (p-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 2 leucines ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCT GTGCTGGCTGGTCTTCTGGGAGCCTGCCAGGCACACCCCATCCCTGAC TCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTC TACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGA GGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCC TGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCA AGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGAT CGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTG AGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGC ACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGA CCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAG CCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGAC CCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCT TCCTGA
12 The nucleotide sequence (L-shaped formula) of FGF-21 without targeting sequencing CACCCCATCC CTGACTCCAG TCCTCTCCTG CAATTCGGGG GCCAAGTCCG GCAGCGGTACCTCTACACAG ATGATGCCCA GCAGACAGAA GCCCACCTGG AGATCAGGGA GGATGGGACGGTGGGGGGCG CTGCTGACCA GAGCCCCGAA AGTCTCCTGC AGCTGAAAGC CTTGAAGCCGGGAGTTATTC AAATCTTGGG AGTCAAGACA TCCAGGTTCC TGTGCCAGCG GCCAGATGGGGCCCTGTATG GATCGCTCCA CTTTGACCCT GAGGCCTGCA GCTTCCGGGA GCTGCTTCTTGAGGACGGAT ACAATGTTTA CCAGTCCGAA GCCCACGGCC TCCCGCTGCA CCTGCCAGGGAACAAGTCCC CACACCGGGA CCCTGCACCC CGAGGACCAG CTCGCTTCCT GCCACTACCAGGCCTGCCCC CCGCACTCCC GGAGCCACCC GGAATCCTGG CCCCCCAGCC CCCCGATGTGGGCTCCTCGG ACCCTCTGAG CATGGTGGGA CCTTCCCAGG GCCGAAGCCCAGCTACGCTTCCTGA
13 The nucleotide sequence (L-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 3 leucines ATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TCA GGA CTG TGG GTT TCT GTG CTG GCT GGT CTT CTG CTG GGA GCC TGC CAG GCA CAC CCC ATC CCT GAC TCC AGT CCT CTC CTG CAA TTC GGG GGC
CAA GTC CGG CAG CGG TAC CTC TAC ACA GAT GAT GCC CAG CAG ACA GAA GCC CAC CTG GAG ATC AGG GAG GAT GGG ACG GTG GGG GGC GCT GCT GAC CAG AGC CCC GAA AGT CTC CTG CAG CTG AAA GCC TTG AAG CCG GGA GTT ATT CAA ATC TTG GGA GTC AAG ACA TCC AGG TTC CTG TGC CAG CGG CCA GAT GGG GCC CTG TAT GGA TCG CTC CAC TTT GAC CCT GAG GCC TGC AGC TTC CGG GAG CTG CTT CTT GAG GAC GGA TAC AAT GTT TAC CAG TCC GAA GCC CAC GGC CTC CCG CTG CAC CTG CCA GGG AAC AAG TCC CCA CAC CGG GAC CCT GCA CCC CGA GGA CCA GCT CGC TTC CTG CCA CTA CCA GGC CTG CCC CCC GCA CTC CCG GAG CCA CCC GGA ATC CTG GCC CCC CAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGC ATG GTG GGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA
14 The nucleotide sequence (L-shaped formula) of FGF-21 with the targeting sequencing-targeting sequencing with 2 leucines ATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TCA GGA CTG TGG GTT TCT GTG CTG GCT GGT CTT CTG GGA GCC TGC CAG GCA CAC CCC ATC CCT GAC TCC AGT CCT CTC CTG CAA TTC GGG GGC CAA GTC CGG CAG CGG TAC CTC TAC ACA GAT GAT GCC CAG CAG ACA GAA GCC CAC CTG GAG ATC AGG GAG GAT GGG ACG GTG GGG GGC GCT GCT GAC CAG AGC CCC GAA AGT CTC CTG CAG CTG AAA GCC TTG AAG CCG GGA GTT ATT CAA ATC TTG GGA GTC AAG ACA TCC AGG TTC CTG TGC CAG CGG CCA GAT GGG GCC CTG TAT GGA TCG CTC CAC TTT GAC CCT GAG GCC TGC AGC TTC CGG GAG CTG CTT CTT GAG GAC GGA TAC AAT GTT TAC CAG TCC GAA GCC CAC GGC CTC CCG CTG CAC CTG CCA GGG AAC AAG TCC CCA CAC CGG GAC CCT GCA CCC CGA GGA CCA GCT CGC TTC CTG CCA CTA CCA GGC CTG CCC CCC GCA CTC CCG GAG CCA CCC GGA ATC CTG GCC CCC CAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGC ATG GTG GGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA
34 FGF-21 amino acid sequence (Rattus norvegicus-ref | NP_570108.1 | [18543365]) Met Asp Trp Met Lys Ser Arg Val Gly Ala Pro Gly Leu Trp Val Cys Leu Leu Leu Pro Val Phe Leu Leu Gly Val Cys Glu Ala Tyr Pro Ile Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Val Gly Thr Ala His Arg Ser Pro Glu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Thr Leu Tyr Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Lys Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu Arg Leu Pro Gln Lys Asp Ser Gln Asp Pro Ala Thr Arg Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Pro His Glu Pro Gln Glu Gln Pro Gly Val Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr Ala Ser
35 FGF-21 amino acid sequence (house mouse-ref | NP_064397.1 | [9910218]) Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly Leu Trp Val Arg Leu Leu Leu Ala Val Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Asp
Gln Asp Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Val Gly Ala Ala His Arg Ser Pro Glu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr Ala Ser
36 FGF-21 amino acid sequence (zebra fish-ref | NP_001038789.1 | [113671792]) Met Leu Phe Ala Cys Phe Phe Ile Phe Phe Ala Leu Phe Pro His Leu Arg Trp Cys Met Tyr Val Pro Ala Gln Asn Val Leu Leu Gln Phe Gly Thr Gln Val Arg Glu Arg Leu Leu Tyr Thr Asp Gly Leu Phe Leu Glu Met Asn Pro Asp Gly Ser Val Lys Gly Ser Pro Glu Lys Asn Leu Asn Cys Val Leu Glu Leu Arg Ser Val Lys Ala Gly Glu Thr Val Ile Gln Ser Ala Ala Thr Ser Leu Tyr Leu Cys Val Asp Asp Gln Asp Lys Leu Lys Gly Gln His His Tyr Ser Ala Leu Asp Cys Thr Phe Gln Glu Leu Leu Leu Asp Gly Tyr Ser Phe Phe Leu Ser Pro His ThrAsn Leu Pro Val Ser Leu Leu Ser Lys Arg Gln Lys His Gly Asn Pro Leu Ser Arg Phe Leu Pro Val Ser Arg Ala Glu Asp Ser Arg Thr Gln Glu Val Lys Gln Tyr Ile Gln Asp Ile Asn Leu Asp Ser Asp Asp Pro Leu Gly Met Gly His Arg Ser His Leu Gln Thr Val Phe Ser Pro Ser Leu His Thr Lys Lys
37 Klotho beta amino acid sequence (homo sapiens-ref | NP_783864.1 | [28376633]) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAVT GFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWK KDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSI SWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEIVTLYHWDLPLALQE KYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTG MHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLG SHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYY KQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHL YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYR FALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHA HLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRL SDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHAD WAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKH RRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSD RDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQ ALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFF TSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPL IFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS
38 Klotho beta amino acid sequence (house mouse-refNP_112457.1GI：13626032) MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRA VTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEG SWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSF YQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDL PLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAW HGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKG WLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEF MKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLN LRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQ AIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSS AHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFT DPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKV THYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYH PTHSHLGLPLPLLS SGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINE PNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLS LHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYI ASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNR SVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITAN GIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKP RFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLV EKKPLIFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQ NIPLKKGHSRVFS
39 OmpA Nucleotide leader sequences atgaaaaaaactgctatcgcgatcgctgtagctctggctggtttcgcgaccgtagctaacgct
40 OmpA amino acid leader sequences M K K T A I A I A V A L A G F A T V A N A
41 MalE Nucleotide leader sequences atgaaaataaaaacaggtgcacgcatcctcgcattatccgcattaacgacgatgatgttttccgcctcggctctcgcc
42 MalE amino acid leader sequences M K I K T G A R I L A L S A L T T M M F S A S A L A
43 StII Nucleotide leader sequences atgaaaaagaatatcgcatttcttcttgcatctatgttcgttttttctattgctacaaatgcctatgca
44 StII amino acid leader sequences M K K N I A F L L A S M F V F S I A T N A Y A
Sequence table
<110>Cujec, Thomas P.
Mariani, Roberto
Hays Putnam, Anna-Maria A.
Keefe, William M.
Knudsen, Nick
Ho, Lillian
Pinkstaff, Jason
Kraynov, Vadim
<120>Modified FGF-21 polypeptides and its purposes
<130>AMBX-0134.OOPCT
<150>60/921,297
<151>2007-03-30
<150>60/988,060
<151>2007-11-14
<160>44
<170>PatentIn version 3.4
<210>1
<211>181
<212>PRT
<213>Homo sapiens
<400>1
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Ala Ser
180
<210>2
<211>191
<212>PRT
<213>Homo sapiens
<400>2
Met His His His His His His Ser Gly Gly His Pro Ile Pro Asp Ser
1 5 10 15
Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr
20 25 30
Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp
35 40 45
Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln
50 55 60
Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr
65 70 75 80
Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu
85 90 95
His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp
100 105 110
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu
115 120 125
Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala
130 135 140
Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro
145 150 155 160
Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu
165 170 175
Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
180 185 190
<210>3
<211>209
<212>PRT
<213>Homo sapiens
<400>3
Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser
1 5 10 15
Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro
20 25 30
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr
35 40 45
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg
50 55 60
Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu
65 70 75 80
Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val
85 90 95
Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly
100 105 110
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu
115 120 125
Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu
130 135 140
His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly
145 150 155 160
Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu
165 170 175
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp
180 185 190
Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
195 200 205
Ser
<210>4
<211>208
<212>PRT
<213>Homo sapiens
<400>4
Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser
1 5 10 15
Val Leu Ala Gly Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp
20 25 30
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu
35 40 45
Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu
50 55 60
Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu
65 70 75 80
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys
85 90 95
Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser
100 105 110
Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu
115 120 125
Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His
130 135 140
Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro
145 150 155 160
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro
165 170 175
Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro
180 185 190
Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
195 200 205
<210>5
<211>181
<212>PRT
<213>Homo sapiens
<400>5
His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val
1 5 10 15
Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His
20 25 30
Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45
Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln
50 55 60
Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly
65 70 75 80
Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg
85 90 95
Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His
100 105 110
Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro
115 120 125
Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro
130 135 140
Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val
145 150 155 160
Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
165 170 175
Pro Ser Tyr Ala Ser
180
<210>6
<211>209
<212>PRT
<213>Homo sapiens
<400>6
Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser
1 5 10 15
Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro
20 25 30
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr
35 40 45
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg
50 55 60
Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu
65 70 75 80
Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val
85 90 95
Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly
100 105 110
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu
115 120 125
Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu
130 135 140
His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly
145 150 155 160
Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu
165 170 175
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp
180 185 190
Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
195 200 205
Ser
<210>7
<211>208
<212>PRT
<213>Homo sapiens
<400>7
Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser
1 5 10 15
Val Leu Ala Gly Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro Asp
20 25 30
Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu
35 40 45
Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu
50 55 60
Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu
65 70 75 80
Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys
85 90 95
Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser
100 105 110
Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu
115 120 125
Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His
130 135 140
Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro
145 150 155 160
Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro
165 170 175
Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro
180 185 190
Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser
195 200 205
<210>8
<211>546
<212>DNA
<213>Homo sapiens
<400>8
caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60
ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 120
gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 180
ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240
gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 300
gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360
aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 420
ggcctgcccc ccgcaccccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 480
ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 540
tcctga 546
<210>9
<211>576
<212>DNA
<213>Homo sapiens
<400>9
atgcatcatc atcatcatca tagcggcggc caccccatcc ctgactccag tcctctcctg 60
caattcgggg gccaagtccg gcagcggtac ctctacacag atgatgccca gcagacagaa 120
gcccacctgg agatcaggga ggatgggacg gtggggggcg ctgctgacca gagccccgaa 180
agtctcctgc agctgaaagc cttgaagccg ggagttattc aaatcttggg agtcaagaca 240
tccaggttcc tgtgccagcg gccagatggg gccctgtatg gatcgctcca ctttgaccct 300
gaggcctgca gcttccggga gctgcttctt gaggacggat acaatgttta ccagtccgaa 360
gcccacggcc tcccgctgca cctgccaggg aacaagtccc cacaccggga ccctgcaccc 420
cgaggaccag ctcgcttcct gccactacca ggcctgcccc ccgcaccccc ggagccaccc 480
ggaatcctgg ccccccagcc ccccgatgtg ggctcctcgg accctctgag catggtggga 540
ccttcccagg gccgaagccc cagctacgct tcctga 576
<210>10
<211>630
<212>DNA
<213>Homo sapiens
<400>10
atggactcgg acgagaccgg gttcgagcac tcaggactgt gggtttctgt gctggctggt 60
cttctgctgg gagcctgcca ggcacacccc atccctgact ccagtcctct cctgcaattc 120
gggggccaag tccggcagcg gtacctctac acagatgatg cccagcagac agaagcccac 180
ctggagatca gggaggatgg gacggtgggg ggcgctgctg accagagccc cgaaagtctc 240
ctgcagctga aagccttgaa gccgggagtt attcaaatct tgggagtcaa gacatccagg 300
ttcctgtgcc agcggccaga tggggccctg tatggatcgc tccactttga ccctgaggcc 360
tgcagcttcc gggagctgct tcttgaggac ggatacaatg tttaccagtc cgaagcccac 420
ggcctcccgc tgcacctgcc agggaacaag tccccacacc gggaccctgc accccgagga 480
ccagctcgct tcctgccact accaggcctg ccccccgcac ccccggagcc acccggaatc 540
ctggcccccc agccccccga tgtgggctcc tcggaccctc tgagcatggt gggaccttcc 600
cagggccgaa gccccagcta cgcttcctga 630
<210>11
<211>627
<212>DNA
<213>Homo sapiens
<400>11
atggactcgg acgagaccgg gttcgagcac tcaggactgt gggtttctgt gctggctggt 60
cttctgggag cctgccaggc acaccccatc cctgactcca gtcctctcct gcaattcggg 120
ggccaagtcc ggcagcggta cctctacaca gatgatgccc agcagacaga agcccacctg 180
gagatcaggg aggatgggac ggtggggggc gctgctgacc agagccccga aagtctcctg 240
cagctgaaag ccttgaagcc gggagttatt caaatcttgg gagtcaagac atccaggttc 300
ctgtgccagc ggccagatgg ggccctgtat ggatcgctcc actttgaccc tgaggcctgc 360
agcttccggg agctgcttct tgaggacgga tacaatgttt accagtccga agcccacggc 420
ctcccgctgc acctgccagg gaacaagtcc ccacaccggg accctgcacc ccgaggacca 480
gctcgcttcc tgccactacc aggcctgccc cccgcacccc cggagccacc cggaatcctg 540
gccccccagc cccccgatgt gggctcctcg gaccctctga gcatggtggg accttcccag 600
ggccgaagcc ccagctacgc ttcctga 627
<210>12
<211>545
<212>DNA
<213>Homo sapiens
<400>12
caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60
ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 120
gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 180
ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240
gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 300
gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360
aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 420
ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 480
ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc agctacgctt 540
cctga 545
<210>13
<211>630
<212>DNA
<213>Homo sapiens
<400>13
atggactcgg acgagaccgg gttcgagcac tcaggactgt gggtttctgt gctggctggt 60
cttctgctgg gagcctgcca ggcacacccc atccctgact ccagtcctct cctgcaattc 120
gggggccaag tccggcagcg gtacctctac acagatgatg cccagcagac agaagcccac 180
ctggagatca gggaggatgg gacggtgggg ggcgctgctg accagagccc cgaaagtctc 240
ctgcagctga aagccttgaa gccgggagtt attcaaatct tgggagtcaa gacatccagg 300
ttcctgtgcc agcggccaga tggggccctg tatggatcgc tccactttga ccctgaggcc 360
tgcagcttcc gggagctgct tcttgaggac ggatacaatg tttaccagtc cgaagcccac 420
ggcctcccgc tgcacctgcc agggaacaag tccccacacc gggaccctgc accccgagga 480
ccagctcgct tcctgccact accaggcctg ccccccgcac tcccggagcc acccggaatc 540
ctggcccccc agccccccga tgtgggctcc tcggaccctc tgagcatggt gggaccttcc 600
cagggccgaa gccccagcta cgcttcctga 630
<210>14
<211>627
<212>DNA
<213>Homo sapiens
<400>14
atggactcgg acgagaccgg gttcgagcac tcaggactgt gggtttctgt gctggctggt 60
cttctgggag cctgccaggc acaccccatc cctgactcca gtcctctcct gcaattcggg 120
ggccaagtcc ggcagcggta cctctacaca gatgatgccc agcagacaga agcccacctg 180
gagatcaggg aggatgggac ggtggggggc gctgctgacc agagccccga aagtctcctg 240
cagctgaaag ccttgaagcc gggagttatt caaatcttgg gagtcaagac atccaggttc 300
ctgtgccagc ggccagatgg ggccctgtat ggatcgctcc actttgaccc tgaggcctgc 360
agcttccggg agctgcttct tgaggacgga tacaatgttt accagtccga agcccacggc 420
ctcccgctgc acctgccagg gaacaagtcc ccacaccggg accctgcacc ccgaggacca 480
gctcgcttcc tgccactacc aggcctgccc cccgcactcc cggagccacc cggaatcctg 540
gccccccagc cccccgatgt gggctcctcg gaccctctga gcatggtggg accttcccag 600
ggccgaagcc ccagctacgc ttcctga 627
<210>15
<211>77
<212>DNA
<213>Manually
<220>
<223>Mutation tRNA derived from Methanococcus jannaschii tRNA
<400>15
ccggcggtag ttcagcaggg cagaacggcg gactctaaat ccgcatggcg ctggttcaaa 60
tccagcccgc cggacca 77
<210>16
<211>306
<212>PRT
<213>Manually
<220>
<223>Mutation synzyme derived from Methanococcus jannaschii synzyme
<400>16
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Set Ala Val
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Tyr Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu His Gly Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Ile His
145 150 155 160
Tyr Glu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>17
<211>77
<212>DNA
<213>Methanococcus jannaschii
<400>17
ccggcggtag ttcagcaggg cagaacggcg gactctaaat ccgcatggcg ctggttcaaa 60
tccggcccgc cggacca 77
<210>18
<211>88
<212>DNA
<213>Manually
<220>
<223>Optimize amber suppressor tRNA
<400>18
cccagggtag ccaagctcgg ccaacggcga cggactctaa atccgttctc gtaggagttc 60
gagggttcga atcccttccc tgggacca 88
<210>19
<211>89
<212>DNA
<213>Manually
<220>
<223>Optimize AGGA frameshift suppressors tRNA
<400>19
gcgagggtag ccaagctcgg ccaacggcga cggacttcct aatccgttct cgtaggagtt 60
cgagggttcg aatccctccc ctcgcacca 89
<210>20
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to azido-L-phenylalanine
<400>20
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr
145 150 155 160
Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>21
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to benzoyl-L-phenylalanine
<400>21
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Ser His
145 150 155 160
Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>22
<211>305
<212>PRT
<213>Manually
<220>
<223>Aminoacyl tRNA synthetase for being incorporated to propargyl-phenylalanine
<400>22
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ala Ile Tyr
145 150 155 160
Leu Ala Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His
165 170 175
Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn
180 185 190
Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys
195 200 205
Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys
210 215 220
Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile
225 230 235 240
Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg
245 250 255
Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu
260 265 270
Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn
275 280 285
Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg
290 295 300
Leu
305
<210>23
<211>305
<212>PRT
<213>Manually
<220>
<223>Aminoacyl tRNA synthetase for being incorporated to propargyl-phenylalanine
<400>23
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ile Pro Tyr
145 150 155 160
Leu Pro Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His
165 170 175
Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn
180 185 190
Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys
195 200 205
Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys
210 215 220
Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile
225 230 235 240
Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg
245 250 255
Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu
260 265 270
Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn
275 280 285
Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg
290 295 300
Leu
305
<210>24
<211>305
<212>PRT
<213>Manually
<220>
<223>Aminoacyl tRNA synthetase for being incorporated to propargyl-phenylalanine
<400>24
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Lys Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ala Ile Tyr
145 150 155 160
Leu Ala Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His
165 170 175
Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn
180 185 190
Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys
195 200 205
Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys
210 215 220
Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile
225 230 235 240
Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg
245 250 255
Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu
260 265 270
Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn
275 280 285
Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg
290 295 300
Leu
305
<210>25
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine
<400>25
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His
145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>26
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine
<400>26
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His
145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>27
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine
<400>27
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Val His
145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>28
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine
<400>28
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Ser His
145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>29
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to acetyl-phenylalanine
<400>29
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Cys His
145 150 155 160
Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>30
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to acetyl-phenylalanine
<400>30
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Thr His
145 150 155 160
Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Vla Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>31
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to acetyl-phenylalanine
<400>31
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Gly His
145 150 155 160
Tyr Leu Gly Val Asp Val Ile Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>32
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to acetyl-phenylalanine
<400>32
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Arg Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Val Ile His
145 150 155 160
Tyr Asp Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile ALa Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>33
<211>306
<212>PRT
<213>Manually
<220>
<223>For being incorporated to the aminoacyl tRNA synthetase to azido-phenylalanine
<400>33
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr
145 150 155 160
Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210>34
<211>208
<212>PRT
<213>Rattus norvegicus
<400>34
Met Asp Trp Met Lys Ser Arg Val Gly Ala Pro Gly Leu Trp Val Cys
1 5 10 15
Leu Leu Leu Pro Val Phe Leu Leu Gly Val Cys Glu Ala Tyr Pro Ile
20 25 30
Ser Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg
35 40 45
Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile
50 55 60
Arg Glu Asp Gly Thr Val Val Gly Thr Ala His Arg Ser Pro Glu Ser
65 70 75 80
Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly
85 90 95
Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Thr Leu Tyr
100 105 110
Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu
115 120 125
Leu Lys Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro
130 135 140
Leu Arg Leu Pro Gln Lys Asp Ser Gln Aso Pro Ala Thr Arg Gly Pro
145 150 155 160
Val Arg Phe Leu Pro Met Pro Gly Leu Pro His Glu Pro Gln Glu Gln
165 170 175
Pro Gly Val Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser Asp Pro
180 185 190
Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr Ala Ser
195 200 205
<210>35
<211>210
<212>PRT
<213>House mouse
<400>35
Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly Leu Trp Val Arg
1 5 10 15
Leu Leu Leu Ala Val Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile
20 25 30
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg
35 40 45
Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile
50 55 60
Arg Glu Asp Gly Thr Val Val Gly Ala Ala His Arg Ser Pro Glu Ser
65 70 75 80
Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly
85 90 95
Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr
100 105 110
Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu
115 120 125
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro
130 135 140
Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp
145 150 155 160
Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln
165 170 175
Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser
180 185 190
Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr
195 200 205
Ala Ser
210
<210>36
<211>194
<212>PRT
<213>Zebra fish
<400>36
Met Leu Phe Ala Cys Phe Phe Ile Phe Phe Ala Leu Phe Pro His Leu
1 5 10 15
Arg Trp Cys Met Tyr Val Pro Ala Gln Asn Val Leu Leu Gln Phe Gly
20 25 30
Thr Gln Val Arg Glu Arg Leu Leu Tyr Thr Asp Gly Leu Phe Leu Glu
35 40 45
Met Asn Pro Asp Gly Ser Val Lys Gly Ser Pro Glu Lys Asn Leu Asn
50 55 60
Cys Val Leu Glu Leu Arg Ser Val Lys Ala Gly Glu Thr Val Ile Gln
65 70 75 80
Ser Ala Ala Thr Ser Leu Tyr Leu Cys Val Asp Asp Gln Asp Lys Leu
85 90 95
Lys Gly Gln His His Tyr Ser Ala Leu Asp Cys Thr Phe Gln Glu Leu
100 105 110
Leu Leu Asp Gly Tyr Ser Phe Phe Leu Ser Pro His Thr Asn Leu Pro
115 120 125
Val Ser Leu Leu Ser Lys Arg Gln Lys His Gly Asn Pro Leu Ser Arg
130 135 140
Phe Leu Pro Val Ser Arg Ala Glu Asp Ser Arg Thr Gln Glu Val Lys
145 150 155 160
Gln Tyr Ile Gln Asp Ile Asn Leu Asp Ser Asp Asp Pro Leu Gly Met
165 170 175
Gly His Arg Ser His Leu Gln Thr Val Phe Ser Pro Ser Leu His Thr
180 185 190
Lys Lys
<210>37
<211>1043
<212>PRT
<213>Homo sapiens
<400>37
Met Lys Pro Gly Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe
1 5 10 15
Phe Ser Thr Asp Glu Ile Thr Thr Arg Tyr Arg Asn Thr Met Ser Asn
20 25 30
Gly Gly Leu Gln Arg Ser Val Ile Leu Ser Ala Leu Ile Leu Leu Arg
35 40 45
Ala Val Thr Gly Phe Ser Gly Asp Gly Arg Ala Ile Trp Ser Lys Asn
50 55 60
Pro Asn Phe Thr Pro Val Asn Glu Ser Gln Leu Phe Leu Tyr Asp Thr
65 70 75 80
Phe Pro Lys Asn Phe Phe Trp Gly Ile Gly Thr Gly Ala Leu Gln Val
85 90 95
Glu Gly Ser Trp Lys Lys Asp Gly Lys Gly Pro Ser Ile Trp Asp His
100 105 110
Phe Ile His Thr His Leu Lys Asn Val Ser Ser Thr Asn Gly Ser Ser
115 120 125
Asp Ser Tyr Ile Phe Leu Glu Lys Asp Leu Ser Ala Leu Asp Phe Ile
130 135 140
Gly Val Ser Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro
145 150 155 160
Asp Gly Ile Val Thr Val Ala Asn Ala Lys Gly Leu Gln Tyr Tyr Ser
165 170 175
Thr Leu Leu Asp Ala Leu Val Leu Arg Asn Ile Glu Ile Val Thr Leu
180 185 190
Tyr His Trp Asp Leu Pro Leu Ala Leu Gln Glu Lys Tyr Gly Gly Trp
195 200 205
Lys Asn Asp Thr Ile Ile Asp Ile Phe Asn Asp Tyr Ala Thr Tyr Cys
210 215 220
Phe Gln Met Phe Gly Asp Arg Val Lys Tyr Trp Ile Thr Ile His Asn
225 230 235 240
Pro Tyr Leu Val Ala Trp His Gly Tyr Gly Thr Gly Met His Ala Pro
245 250 255
Gly Glu Lys Gly Asn Leu Ala Ala Val Tyr Thr Val Gly His Asn Leu
260 265 270
Ile Lys Ala His Ser Lys Val Trp His Asn Tyr Asn Thr His Phe Arg
275 280 285
Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp Ile
290 295 300
Glu Pro Asn Arg Ser Glu Asn Thr Met Asp Ile Phe Lys Cys Gln Gln
305 310 315 320
Ser Met Val Ser Val Leu Gly Trp Phe Ala Asn Pro Ile His Gly Asp
325 330 335
Gly Asp Tyr Pro Glu Gly Met Arg Lys Lys Leu Phe Ser Val Leu Pro
340 345 350
Ile Phe Ser Glu Ala Glu Lys His Glu Met Arg Gly Thr Ala Asp Phe
355 360 365
Phe Ala Phe Ser Phe Gly Pro Asn Asn Phe Lys Pro Leu Asn Thr Met
370 375 380
Ala Lys Met Gly Gln Asn Val Ser Leu Asn Leu Arg Glu Ala Leu Asn
385 390 395 400
Trp Ile Lys Leu Glu Tyr Asn Asn Pro Arg Ile Leu Ile Ala Glu Asn
405 410 415
Gly Trp Phe Thr Asp Ser Arg Val Lys Thr Glu Asp Thr Thr Ala Ile
420 425 430
Tyr Met Met Lys Asn Phe Leu Ser Gln Val Leu Gln Ala Ile Arg Leu
435 440 445
Asp Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp Ser Leu Leu Asp Gly
450 455 460
Phe Glu Trp Gln Asp Ala Tyr Thr Ile Arg Arg Gly Leu Phe Tyr Val
465 470 475 480
Asp Phe Asn Ser Lys Gln Lys Glu Arg Lys Pro Lys Ser Ser Ala His
485 490 495
Tyr Tyr Lys Gln Ile Ile Arg Glu Asn Gly Phe Ser Leu Lys Glu Ser
500 505 510
Thr Pro Asp Val Gln Gly Gln Phe Pro Cys Asp Phe Ser Trp Gly Val
515 520 525
Thr Glu Ser Val Leu Lys Pro Glu Ser Val Ala Ser Ser Pro Gln Phe
530 535 540
Ser Asp Pro His Leu Tyr Val Trp Asn Ala Thr Gly Asn Arg Leu Leu
545 550 555 560
His Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro Ala Gln Cys Thr
565 570 575
Asp Phe Val Asn Ile Lys Lys Gln Leu Glu Met Leu Ala Arg Met Lys
580 585 590
Val Thr His Tyr Arg Phe Ala Leu Asp Trp Ala Ser Val Leu Pro Thr
595 600 605
Gly Asn Leu Ser Ala Val Asn Arg Gln Ala Leu Arg Tyr Tyr Arg Cys
610 615 620
Val Val Ser Glu Gly Leu Lys Leu Gly Ile Ser Ala Met Val Thr Leu
625 630 635 640
Tyr Tyr Pro Thr His Ala His Leu Gly Leu Pro Glu Pro Leu Leu His
645 650 655
Ala Asp Gly Trp Leu Asn Pro Ser Thr Ala Glu Ala Phe Gln Ala Tyr
660 665 670
Ala Gly Leu Cys Phe Gln Glu Leu Gly Asp Leu Val Lys Leu Trp Ile
675 680 685
Thr Ile Asn Glu Pro Asn Arg Leu Ser Asp Ile Tyr Asn Arg Ser Gly
690 695 700
Asn Asp Thr Tyr Gly Ala Ala His Asn Leu Leu Val Ala His Ala Leu
705 710 715 720
Ala Trp Arg Leu Tyr Asp Arg Gln Phe Arg Pro Ser Gln Arg Gly Ala
725 730 735
Val Ser Leu Ser Leu His Ala Asp Trp Ala Glu Pro Ala Asn Pro Tyr
740 745 750
Ala Asp Ser His Trp Arg Ala Ala Glu Arg Phe Leu Gln Phe Glu Ile
755 760 765
Ala Trp Phe Ala Glu Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ala Ala
770 775 780
Met Arg Glu Tyr Ile Ala Ser Lys His Arg Arg Gly Leu Ser Ser Ser
785 790 795 800
Ala Leu Pro Arg Leu Thr Glu Ala Glu Arg Arg Leu Leu Lys Gly Thr
805 810 815
Val Asp Phe Cys Ala Leu Asn His Phe Thr Thr Arg Phe Val Met His
820 825 830
Glu Gln Leu Ala Gly Ser Arg Tyr Asp Ser Asp Arg Asp Ile Gln Phe
835 840 845
Leu Gln Asp Ile Thr Arg Leu Ser Ser Pro Thr Arg Leu Ala Val Ile
850 855 860
Pro Trp Gly Val Arg Lys Leu Leu Arg Trp Val Arg Arg Asn Tyr Gly
865 870 875 880
Asp Met Asp Ile Tyr Ile Thr Ala Ser Gly Ile Asp Asp Gln Ala Leu
885 890 895
Glu Asp Asp Arg Leu Arg Lys Tyr Tyr Leu Gly Lys Tyr Leu Gln Glu
900 905 910
Val Leu Lys Ala Tyr Leu Ile Asp Lys Val Arg Ile Lys Gly Tyr Tyr
915 920 925
Ala Phe Lys Leu Ala Glu Glu Lys Ser Lys Pro Arg Phe Gly Phe Phe
930 935 940
Thr Ser Asp Phe Lys Ala Lys Ser Ser Ile Gln Phe Tyr Asn Lys Val
945 950 955 960
Ile Ser Ser Arg Gly Phe Pro Phe Glu Asn Ser Ser Ser Arg Cys Ser
965 970 975
Gln Thr Gln Glu Asn Thr Glu Cys Thr Val Cys Leu Phe Leu Val Gln
980 985 990
Lys Lys Pro Leu Ile Phe Leu Gly Cys Cys Phe Phe Ser Thr Leu Val
995 1000 1005
Leu Leu Leu Ser Ile Ala Ile Phe Gln Arg Gln Lys Arg Arg Lys
1010 1015 1020
Phe Trp Lys Ala Lys Asn Leu Gln His Ile Pro Leu Lys Lys Gly
1025 1030 1035
Lys Arg Val Val Ser
1040
<210>38
<211>1043
<212>PRT
<213>House mouse
<400>38
Met Lys Thr Gly Cys Ala Ala Gly Ser Pro Gly Asn Glu Trp Ile Phe
1 5 10 15
Phe Ser Ser Asp Glu Arg Asn Thr Arg Ser Arg Lys Thr Met Ser Asn
20 25 30
Arg Ala Leu Gln Arg Ser Ala Val Leu Ser Ala Phe Val Leu Leu Arg
35 40 45
Ala Val Thr Gly Phe Ser Gly Asp Gly Lys Ala Ile Trp Asp Lys Lys
50 55 60
Gln Tyr Val Ser Pro Val Asn Pro Ser Gln Leu Phe Leu Tyr Asp Thr
65 70 75 80
Phe Pro Lys Asn Phe Ser Trp Gly Val Gly Thr Gly Ala Phe Gln Val
85 90 95
Glu Gly Ser Trp Lys Thr Asp Gly Arg Gly Pro Ser Ile Trp Asp Arg
100 105 110
Tyr Val Tyr Ser His Leu Arg Gly Val Asn Gly Thr Asp Arg Ser Thr
115 120 125
Asp Ser Tyr Ile Phe Leu Glu Lys Asp Leu Leu Ala Leu Asp Phe Leu
130 135 140
Gly Val Ser Phe Tyr Gln Phe Ser Ile Ser Trp Pro Arg Leu Phe Pro
145 150 155 160
Asn Gly Thr Val Ala Ala Val Asn Ala Gln Gly Leu Arg Tyr Tyr Arg
165 170 175
Ala Leu Leu Asp Ser Leu Val Leu Arg Asn Ile Glu Pro Ile Val Thr
180 185 190
Leu Tyr His Trp Asp Leu Pro Leu Thr Leu Gln Glu Glu Tyr Gly Gly
195 200 205
Trp Lys Asn Ala Thr Met Ile Asp Leu Phe Asn Asp Tyr Ala Thr Tyr
210 215 220
Cys Phe Gln Thr Phe Gly Asp Arg Val Lys Tyr Trp Ile Thr Ile His
225 230 235 240
Asn Pro Tyr Leu Val Ala Trp His Gly Phe Gly Thr Gly Met His Ala
245 250 255
Pro Gly Glu Lys Gly Asn Leu Thr Ala Val Tyr Thr Val Gly His Asn
260 265 270
Leu Ile Lys Ala His Ser Lys Val Trp His Asn Tyr Asp Lys Asn Phe
275 280 285
Arg Pro His Gln Lys Gly Trp Leu Ser Ile Thr Leu Gly Ser His Trp
290 295 300
Ile Glu Pro Asn Arg Thr Asp Asn Met Glu Asp Val Ile Asn Cys Gln
305 310 315 320
His Ser Met Ser Ser Val Leu Gly Trp Phe Ala Asn Pro Ile His Gly
325 330 335
Asp Gly Asp Tyr Pro Glu Phe Met Lys Thr Gly Ala Met Ile Pro Glu
340 345 350
Phe Ser Glu Ala Glu Lys Glu Glu Val Arg Gly Thr Ala Asp Phe Phe
355 360 365
Ala Phe Ser Phe Gly Pro Asn Asn Phe Arg Pro Ser Asn Thr Val Val
370 375 380
Lys Met Gly Gln Asn Val Ser Leu Asn Leu Arg Gln Val Leu Asn Trp
385 390 395 400
Ile Lys Leu Glu Tyr Asp Asp Pro Gln Ile Leu Ile Ser Glu Asn Gly
405 410 415
Trp Phe Thr Asp Ser Tyr Ile Lys Thr Glu Asp Thr Thr Ala Ile Tyr
420 425 430
Met Met Lys Asn Phe Leu Asn Gln Val Leu Gln Ala Ile Lys Phe Asp
435 440 445
Glu Ile Arg Val Phe Gly Tyr Thr Ala Trp Thr Leu Leu Asp Gly Phe
450 455 460
Glu Trp Gln Asp Ala Tyr Thr Thr Arg Arg Gly Leu Phe Tyr Val Asp
465 470 475 480
Phe Asn Ser Glu Gln Lys Glu Arg Lys Pro Lys Ser Ser Ala His Tyr
485 490 495
Tyr Lys Gln Ile Ile Gln Asp Asn Gly Phe Pro Leu Lys Glu Ser Thr
500 505 510
Pro Asp Met Lys Gly Arg Phe Pro Cys Asp Phe Ser Trp Gly Val Thr
515 520 525
Glu Ser Val Leu Lys Pro Glu Phe Thr Val Ser Ser Pro Gln Phe Thr
530 535 540
Asp Pro His Leu Tyr Val Trp Asn Val Thr Gly Asn Arg Leu Leu Tyr
545 550 555 560
Arg Val Glu Gly Val Arg Leu Lys Thr Arg Pro Ser Gln Cys Thr Asp
565 570 575
Tyr Val Ser Ile Lys Lys Arg Val Glu Met Leu Ala Lys Met Lys Val
580 585 590
Thr His Tyr Gln Phe Ala Leu Asp Trp Thr Ser Ile Leu Pro Thr Gly
595 600 605
Asn Leu Ser Lys Val Asn Arg Gln Val Leu Arg Tyr Tyr Arg Cys Val
610 615 620
Val Ser Glu Gly Leu Lys Leu Gly Val Phe Pro Met Val Thr Leu Tyr
625 630 635 640
His Pro Thr His Ser His Leu Gly Leu Pro Leu Pro Leu Leu Ser Ser
645 650 655
Gly Gly Trp Leu Asn Met Asn Thr Ala Lys Ala Phe Gln Asp Tyr Ala
660 665 670
Glu Leu Cys Phe Arg Glu Leu Gly Asp Leu Val Lys Leu Trp Ile Thr
675 680 685
Ile Asn Glu Pro Asn Arg Leu Ser Asp Met Tyr Asn Arg Thr Ser Asn
690 695 700
Asp Thr Tyr Arg Ala Ala His Asn Leu Met Ile Ala His Ala Gln Val
705 710 715 720
Trp His Leu Tyr Asp Arg Gln Tyr Arg Pro Val Gln His Gly Ala Val
725 730 735
Ser Leu Ser Leu His Cys Asp Trp Ala Glu Pro Ala Asn Pro Phe Val
740 745 750
Asp Ser His Trp Lys Ala Ala Glu Arg Phe Leu Gln Phe Glu Ile Ala
755 760 765
Trp Phe Ala Asp Pro Leu Phe Lys Thr Gly Asp Tyr Pro Ser Val Met
770 775 780
Lys Glu Tyr Ile Ala Ser Lys Asn Gln Arg Gly Leu Ser Ser Ser Val
785 790 795 800
Leu Pro Arg Phe Thr Ala Lys Glu Ser Arg Leu Val Lys Gly Thr Val
805 810 815
Asp Phe Tyr Ala Leu Asn His Phe Thr Thr Arg Phe Val Ile His Lys
820 825 830
Gln Leu Asn Thr Asn Arg Ser Val Ala Asp Arg Asp Val Gln Phe Leu
835 840 845
Gln Asp Ile Thr Arg Leu Ser Ser Pro Ser Arg Leu Ala Val Thr Pro
850 855 860
Trp Gly Val Arg Lys Leu Leu Ala Trp Ile Arg Arg Asn Tyr Arg Asp
865 870 875 880
Arg Asp Ile Tyr Ile Thr Ala Asn Gly Ile Asp Asp Leu Ala Leu Glu
885 890 895
Asp Asp Gln Ile Arg Lys Tyr Tyr Leu Glu Lys Tyr Val Gln Glu Ala
900 905 910
Leu Lys Ala Tyr Leu Ile Asp Lys Val Lys Ile Lys Gly Tyr Tyr Ala
915 920 925
Phe Lys Leu Thr Glu Glu Lys Ser Lys Pro Arg Phe Gly Phe Phe Thr
930 935 940
Ser Asp Phe Arg Ala Lys Ser Ser Val Gln Phe Tyr Ser Lys Leu Ile
945 950 955 960
Ser Ser Ser Gly Leu Pro Ala Glu Asn Arg Ser Pro Ala Cys Gly Gln
965 970 975
Pro Ala Glu Asp Thr Asp Cys Thr Ile Cys Ser Phe Leu Val Glu Lys
980 985 990
Lys Pro Leu Ile Phe Phe Gly Cys Cys Phe Ile Ser Thr Leu Ala Val
995 1000 1005
Leu Leu Ser Ile Thr Val Phe His His Gln Lys Arg Arg Lys Phe
1010 1015 1020
Gln Lys Ala Arg Asn Leu Gln Asn Ile Pro Leu Lys Lys Gly His
1025 1030 1035
Ser Arg Val Phe Ser
1040
<210>39
<211>63
<212>DNA
<213>Manually
<220>
<223>It is cloned into the FGF21 secretion constructs in pVK7ara (Nde/Eco)
<400>39
atgaaaaaaa ctgctatcgc gatcgctgta gctctggctg gtttcgcgac cgtagctaac 60
gct 63
<210>40
<211>21
<212>PRT
<213>Manually
<220>
<223>It is cloned into the FGF21 secretion constructs in pVK7ara (Nde/Eco)
<400>40
Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala
1 5 10 15
Thr Val Ala Asn Ala
20
<210>41
<211>78
<212>DNA
<213>Manually
<220>
<223>It is cloned into the FGF21 secretion constructs in pVK7ara (Nde/Eco)
<400>41
atgaaaataa aaacaggtgc acgcatcctc gcattatccg cattaacgac gatgatgttt 60
tccgcctcgg ctctcgcc 78
<210>42
<211>26
<212>PRT
<213>Manually
<220>
<223>It is cloned into the FGF21 secretion constructs in pVK7ara (Nde/Eco)
<400>42
Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15
Thr Met Met Phe Ser Ala Ser Ala Leu Ala
20 25
<210>43
<211>69
<212>DNA
<213>Manually
<220>
<223>It is cloned into the FGF21 secretion constructs in pVK7ara (Nde/Eco)
<400>43
atgaaaaaga atatcgcatt tcttcttgca tctatgttcg ttttttctat tgctacaaat 60
gcctatgca 69
<210>44
<211>23
<212>PRT
<213>Manually
<220>
<223>It is cloned into the FGF21 secretion constructs in pVK7ara (Nde/Eco)
<400>44
Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe Ser
1 5 10 15
Ile Ala Thr Asn Ala Tyr Ala
20

Claims (54)

1. a kind of through modifying mankind's FGF-21 polypeptides, it is by the SEQ ID NO with the methionine for being blended in N-terminal:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4th, the SEQ ID NO with the methionine for being blended in N-terminal:5、SEQ ID NO:6 or SEQ ID NO:7 peptide sequence is constituted, wherein in SEQ ID NO:At 1 108 positions or the SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6 or SEQ ID NO:In 7 peptide sequence Corresponding amino acid position replaced with a non-naturally encoded amino acids, and the peptide sequence further includes most days Right coded amino acid substitution, amino acid deletions, an amino acid adding or a conservative replaces；Wherein described non-natural Coded amino acid is connected with water-soluble polymer, and the water-soluble polymer includes polyethylene glycol or oligosaccharides；It is described through modification Mankind's FGF-21 polypeptides have at least one FGF-21 bioactivity, and the non-naturally encoded amino acids are selected from contraposition and replaced Phenylalanine.
2. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the non-naturally encoded amino acids include carbonyl Base, aminooxy group, diazanyl, hydrazide group, amino urea groups, azido or alkynyl.
3. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the non-naturally encoded amino acids are to second Acylphenylalanines.
4. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the non-naturally encoded amino acids are comprising p- Azido-phenylalanine.
5. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the mankind FGF-21 polypeptides are by 108 The SEQ ID NO with the methionine for being blended in N-terminal through a non-naturally encoded amino acids substitution at position:1 is constituted.
6. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the mankind FGF-21 polypeptides are by 108 Through a SEQ ID NO with the methionine for being blended in N-terminal replaced to acetyl phenyl alanine at position:1 is constituted.
7. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the water-soluble polymer is polyethylene glycol, And with the mean molecule quantity between 0.1kDa and 100kDa.
8. a kind of through modifying mankind's FGF-21 polypeptides, it is made up of following：
SEQ ID NO with the methionine for being blended in N-terminal:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、 SEQ ID NO with the methionine for being blended in N-terminal:5、SEQ ID NO:6 or SEQ ID NO:7 peptide sequence, wherein In SEQ ID NO:At 1 108 positions or the SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO: 5、SEQ ID NO:6 or SEQ ID NO:Corresponding amino acid position in 7 peptide sequence is with a non-naturally encoded amino acids Replaced；Wherein described non-naturally encoded amino acids are connected with water-soluble polymer, and the water-soluble polymer is by gathering Ethylene glycol or oligosaccharides are constituted；It is described that there is at least one FGF-21 bioactivity, and its through modifying mankind's FGF-21 polypeptides Described in non-naturally encoded amino acids be to acetyl phenyl alanine.
9. it is according to claim 8 through modify mankind's FGF-21 polypeptides, wherein the water-soluble polymer be have between The polyethylene glycol of mean molecule quantity between 1kDa and 100kDa.
10. it is according to claim 1 through modify mankind's FGF-21 polypeptides, wherein the non-naturally encoded amino acids be SEQ ID NO:At 1 108 positions or SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6 or SEQ ID NO:Replace at corresponding amino acid position in 7 to acetyl phenyl alanine, and the water-soluble poly Compound is polyethylene glycol.
11. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the water-soluble polymer is polyethylene glycol And the mean molecule quantity with 30kDa.
12. according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the water-soluble polymer be in branch or Multi-arm.
13. it is according to claim 12 through modify mankind's FGF-21 polypeptides, wherein the water-soluble polymer have between Mean molecule quantity between 1kDa and 100kDa.
14. it is according to claim 1 through modifying mankind's FGF-21 polypeptides, wherein the water-soluble polymer is oligosaccharides.
15. a kind of through modifying mankind's FGF-21 polypeptides, it is by with the methionine for being blended in N-terminal and in 108 positions warp The SEQ ID NO of non-naturally encoded amino acids substitution:1 sequence is constituted, wherein the non-naturally encoded amino acids are to second Acylphenylalanines, it is described that acetyl phenyl alanine is connected with water-soluble polymer, wherein the water-soluble polymer is tool There is the polyalkylene glycol moiety of 30kDa mean molecule quantities, wherein described have at least one FGF- through modifying mankind FGF-21 polypeptides 21 bioactivity.
16. according to any one of claim 1 to 15 through modify mankind's FGF-21 polypeptides, wherein it is described through modify the mankind The vivo half-life that FGF-21 polypeptides have, the non-naturally encoded amino acids and the water-soluble poly are not included The corresponding mankind FGF-21 polypeptides of compound are height.
17. it is according to claim 15 through modifying mankind's FGF-21 polypeptides, wherein described through modifying mankind's FGF-21 polypeptides The serum half-life being had, is the corresponding human for not including the non-naturally encoded amino acids and the water-soluble polymer At least five times of the serum half-life of class FGF-21 polypeptides.
18. it is according to claim 17 through modifying mankind's FGF-21 polypeptides, wherein described through modifying mankind's FGF-21 polypeptides The serum half-life being had, is the corresponding human for not including the non-naturally encoded amino acids and the water-soluble polymer At least 10 times of the serum half-life of class FGF-21 polypeptides.
19. a kind of through modifying mankind's FGF-21 polypeptides, it is by the SEQ ID NO with the methionine for being blended in N-terminal:1、 SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4th, the SEQ ID NO with the methionine for being blended in N-terminal:5、SEQ ID NO:6 or SEQ ID NO:7 peptide sequence is constituted, wherein in SEQ ID NO:At 1 108 positions or the SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6 or SEQ ID NO:In 7 peptide sequence Corresponding amino acid position replaced with a non-naturally encoded amino acids, and in SEQ ID NO:At 1 118,134 positions Or in the SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6 or SEQ ID NO:Corresponding amino acid position in 7 peptide sequence replaces leucine and alanine with cysteine respectively, wherein described many Peptide sequence further includes most one natural coded amino acids substitutions, amino acid deletions, an amino acid adding or a guarantor Keeping property replaces；Wherein described non-naturally encoded amino acids are connected with water-soluble polymer, and the water-soluble polymer includes gathering Ethylene glycol or oligosaccharides；It is described that there is at least one FGF-21 bioactivity, and the non-day through modifying mankind's FGF-21 polypeptides Right coded amino acid is selected from the phenylalanine of contraposition substitution.
20. a kind of through modifying mankind's FGF-21 polypeptides, it is by the SEQ ID NO with the methionine for being blended in N-terminal:1、 SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4th, the SEQ ID NO with the methionine for being blended in N-terminal:5、SEQ ID NO:6 or SEQ ID NO:7 peptide sequence is constituted, wherein in SEQ ID NO:At 1 108 positions or the SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6 or SEQ ID NO:In 7 peptide sequence Corresponding amino acid position replaced with a non-naturally encoded amino acids, and in SEQ ID NO:At 1 170,172 positions Or in the SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6 or SEQ ID NO:Corresponding amino acid position in 7 peptide sequence replaces sweet ammonia respectively with natural a coding or non-naturally encoded amino acids Acid and serine, wherein the peptide sequence further include most one natural coded amino acids replace, amino acid deletions, An amino acid adding or a conservative replaces；Wherein described non-naturally encoded amino acids are connected with water-soluble polymer, and The water-soluble polymer includes polyethylene glycol or oligosaccharides；It is described that there is at least one FGF-21 through modifying mankind FGF-21 polypeptides Bioactivity, and the non-naturally encoded amino acids be selected from contraposition substitution phenylalanine.
21. a kind of composition, it is comprising pharmaceutically acceptable supporting agent and according to any in claim 1 to 15,19 and 20 Described in through modify mankind's FGF-21 polypeptides.
22. composition according to claim 21, wherein described composition further includes second medicament.
23. composition according to claim 22, wherein described second medicament is selected from by the following group constituted Group：Biguanides, thiazolidinedione, sulfonylurea, benzoic acid derivative, the mixture of alpha-glucosidase inhibitors and aforementioned substances.
24. a kind of composition, it includes pharmaceutically acceptable supporting agent and according to claim 15 through modifying the mankind FGF-21 polypeptides.
25. a kind of purposes of the composition according to claim 21, can add for manufacturing for being used to treat through administration FGF-21 With the medicament in the therapeutic scheme of the symptom for the treatment of or prevention, wherein the medicament includes the composition according to claim 21 Prevention or therapeutically effective amount.
26. purposes according to claim 25, wherein the therapeutic scheme is according to claim 25 including administration Medicament and administration include the second medicament of medication, and the wherein administration of foregoing agents can be carried out at the same time or separately, can be according to Any order administration.
27. purposes according to claim 26, wherein the medication is selected from by the following group constituted：It is double Guanidine, thiazolidinedione, sulfonylurea, benzoic acid derivative, the mixture of alpha-glucosidase inhibitors and aforementioned substances.
28. a kind of purposes of the composition according to claim 24, can add for manufacturing for being used to treat through administration FGF-21 With the medicament in the therapeutic scheme of the symptom for the treatment of or prevention, wherein the medicament includes the composition according to claim 24 Prevention or therapeutically effective amount.
29. a kind of purposes of the composition according to claim 21, for manufacturing for being used for the Portugal via increase fat cell Grape Sugar intake and increase glucose utilization or treatment, the medicament in the therapeutic scheme that prevents or alleviate following symptom：2 type glycosurias Disease, obesity, insulin resistance, hyperinsulinemia, poor glucose tolerance, hyperglycemia, metabolic syndrome or foregoing disease The combination of disease；Wherein described medicament includes the prevention of composition or therapeutically effective amount according to claim 21.
30. purposes according to claim 29, wherein the therapeutic scheme is according to claim 29 including administration Medicament and administration include the second medicament of medication, and the wherein administration of foregoing agents can be carried out at the same time or separately, can be according to Any order administration.
31. purposes according to claim 30, wherein the medication is selected from by the following group constituted：It is double Guanidine, thiazolidinedione, sulfonylurea, benzoic acid derivative, the mixture of alpha-glucosidase inhibitors and aforementioned substances.
32. a kind of nucleic acid, it includes at least one selection codon, thereby expressed in the case of it there are orthogonal tRNA When, it is allowed to the expression of polypeptides according to claim 1 through modifying mankind's FGF-21 polypeptides can be produced；Wherein described nucleic acid is Coding is described through modifying mankind's FGF-21 polypeptides, and at least one described selection codon is that coding is described through modifying mankind FGF-21 The non-naturally encoded amino acids of polypeptide.
33. a kind of nucleic acid, it includes at least one selection codon, thereby expressed in the case of it there are orthogonal tRNA When, it is allowed to it can produce according to claim 3 through modifying mankind's FGF-21 polypeptides；Wherein described nucleic acid is the coding warp Mankind's FGF-21 polypeptides are modified, at least one described selection codon is the coding non-day through modifying mankind's FGF-21 polypeptides Right coded amino acid.
34. a kind of nucleic acid, it includes at least one selection codon, thereby expressed in the case of it there are orthogonal tRNA When, it is allowed to it can produce according to claim 2 through modifying mankind's FGF-21 polypeptides；Wherein described nucleic acid is the coding warp Mankind's FGF-21 polypeptides are modified, at least one described selection codon is the coding non-day through modifying mankind's FGF-21 polypeptides Right coded amino acid.
35. a kind of nucleic acid, it includes at least one selection codon, thereby expressed in the case of it there are orthogonal tRNA When, it is allowed to it can produce according to claim 5 through modifying mankind's FGF-21 polypeptides；Wherein described nucleic acid is the coding warp Mankind's FGF-21 polypeptides are modified, at least one described selection codon is the coding non-day through modifying mankind's FGF-21 polypeptides Right coded amino acid.
36. a kind of nucleic acid, it includes at least one selection codon, thereby expressed in the case of it there are orthogonal tRNA When, it is allowed to it can produce according to claim 6 through modifying mankind's FGF-21 polypeptides；Wherein described nucleic acid is the coding warp Mankind's FGF-21 polypeptides are modified, at least one described selection codon is the coding non-day through modifying mankind's FGF-21 polypeptides Right coded amino acid.
37. a kind of cell, it includes nucleic acid according to claim 32, wherein the cell includes orthogonal tRNA/synthetase And orthogonal tRNA.
38. a kind of cell, it includes nucleic acid according to claim 33, wherein the cell includes orthogonal tRNA/synthetase And orthogonal tRNA.
39. a kind of cell, it includes nucleic acid according to claim 34, wherein the cell includes orthogonal tRNA/synthetase And orthogonal tRNA.
40. a kind of cell, it includes nucleic acid according to claim 35, wherein the cell includes orthogonal tRNA/synthetase And orthogonal tRNA.
41. a kind of cell, it includes nucleic acid according to claim 36, wherein the cell includes orthogonal tRNA/synthetase And orthogonal tRNA.
42. a kind of produce the method through modifying mankind's FGF-21 polypeptides in eukaryotic, methods described includes：In appropriate training The eukaryotic that culture in base includes the nucleic acid according to claim 32 is supported, wherein when nucleic acid is in including orthogonal tRNA synthesis When being expressed in enzyme and orthogonal tRNA eukaryotic, you can produce according to claim 1 through modifying the mankind FGF-21 polypeptides.
43. a kind of produce the method through modifying mankind's FGF-21 polypeptides in eukaryotic, methods described includes：In appropriate training The eukaryotic that culture in base includes the nucleic acid according to claim 33 is supported, wherein when nucleic acid is in including orthogonal tRNA synthesis When being expressed in enzyme and orthogonal tRNA eukaryotic, you can produce according to claim 3 through modifying the mankind FGF-21 polypeptides.
44. a kind of produce the method through modifying mankind's FGF-21 polypeptides in eukaryotic, methods described includes：In appropriate training The eukaryotic that culture in base includes the nucleic acid according to claim 34 is supported, wherein when nucleic acid is in including orthogonal tRNA synthesis When being expressed in enzyme and orthogonal tRNA eukaryotic, you can produce according to claim 2 through modifying the mankind FGF-21 polypeptides.
45. a kind of produce the method through modifying mankind's FGF-21 polypeptides in eukaryotic, methods described includes：In appropriate training The eukaryotic that culture in base includes the nucleic acid according to claim 35 is supported, wherein when nucleic acid is in including orthogonal tRNA synthesis When being expressed in enzyme and orthogonal tRNA eukaryotic, you can produce according to claim 5 through modifying the mankind FGF-21 polypeptides.
46. a kind of produce the method through modifying mankind's FGF-21 polypeptides in eukaryotic, methods described includes：In appropriate training The eukaryotic that culture in base includes the nucleic acid according to claim 36 is supported, wherein when nucleic acid is in including orthogonal tRNA synthesis When being expressed in enzyme and orthogonal tRNA eukaryotic, you can produce according to claim 6 through modifying the mankind FGF-21 polypeptides.
47. a kind of produce the method through modifying mankind's FGF-21 polypeptides in eukaryotic, methods described includes：In appropriate training Support base in culture comprising according to claim 32 nucleic acid eukaryotic, when nucleic acid in include orthogonal tRNA/synthetase with And when being expressed in orthogonal tRNA eukaryotic, you can produce through modifying mankind's FGF-21 polypeptides；Then make described through repairing The water-soluble polymer adornd mankind FGF-21 polypeptides and can reacted with the non-naturally encoded amino acids reacts, you can produce basis Through modifying mankind's FGF-21 polypeptides described in claim 1, wherein the water-soluble polymer includes polyethylene glycol or oligosaccharides.
48. method according to claim 47, wherein described water-soluble polymer include alkynyl, aminooxy group, azido, Diazanyl, hydrazide group or semicarbazides base section.
49. method according to claim 48, wherein described alkynyl, aminooxy group, azido, diazanyl, hydrazide group or ammonia Base urea groups part is bonded and be connected with water-soluble polymer via acid amides.
50. method according to claim 47, wherein the water-soluble polymer includes polyethylene glycol.
51. method according to claim 50, wherein the polyalkylene glycol moiety has 30kDa mean molecule quantity.
52. method according to claim 48, wherein the non-naturally encoded amino acids are included to acetyl phenyl alanine.
53. method according to claim 48, wherein the FGF-21 polypeptides include with the first sulphur ammonia for being blended in N-terminal Acid and the SEQ ID NO through a non-naturally encoded amino acids substitution at 108 positions:1.
54. method according to claim 48, wherein the FGF-21 polypeptides include with the first sulphur ammonia for being blended in N-terminal Acid and at 108 positions through one to acetyl phenyl alanine replace SEQ ID NO:1.
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