CN101090980A - Modified human growth hormone - Google Patents

Modified human growth hormone Download PDF

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CN101090980A
CN101090980A CNA2005800444641A CN200580044464A CN101090980A CN 101090980 A CN101090980 A CN 101090980A CN A2005800444641 A CNA2005800444641 A CN A2005800444641A CN 200580044464 A CN200580044464 A CN 200580044464A CN 101090980 A CN101090980 A CN 101090980A
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peg
amino acid
hgh
alkylene
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丘霍松
托马斯·O·丹尼尔
理查德·D·迪马什
安娜·玛丽亚·海斯
特洛伊·E·威尔逊
比·查·西姆
戴维·C·利青格
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Ambrx Inc
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Abstract

Modified human growth hormone polypeptides and uses thereof are provided.

Description

Modified human growth hormone
The cross reference of related application
The application's case is advocated the U.S. Provisional Patent Application case the 60/638th of application on December 22nd, 2004, the U.S. Provisional Patent Application case the 60/727th of applying for on October 17th, 2005 for No. 616, No. 996 right of priority is incorporated herein the specification sheets of described application case in full.
Technical field
The present invention relates to through at least one non-naturally encoded amino acid modified growth hormone polypeptides.
Background technology
Tethelin (GH) supergene family (Bazan, F.Immunology Today 11:350-354 (1990); Mott, H.R. and Campbell, I.D.Current Opinion in Structural Biology 5:114-121 (1995); Silvennoinen, O. and Ihle, J.N. (1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINERECEPTORS) represent one group of protein with similar structures feature.Each member in this protein families all comprises a four-helix bundle, shows its general structure in Fig. 1.Though in this family, still have more member to await differentiating that some members wherein comprise following protein: tethelin, prolactin, the placenta lactogen, erythropoietin (EPO), thrombosis element (TPO), IL-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukaemia inhibitory factor, interferon-alpha, interferon-, IFN-, omega interferon, the τ Interferon, rabbit, the ε Interferon, rabbit, granulocyte colony-stimulating factor (G-CSF), rHuGM-CSF (GM-CSF), macrophage colony stimulating factor (M-CSF) and myocardial nutrition element-1 (CT-1) (" GH supergene family ").Although the member of GH supergene family generally has limited amino acid or consensus dna sequence, they have similar secondary and tertiary structure.Total constitutional features is differentiated the newcomer of gene family easily.The general structure that in Fig. 2, Fig. 3, Fig. 4 and Fig. 5, shows family member hGH, EPO, IFN α-2 and G-CSF respectively.
Human growth hormone (hGH) is one of GH supergene family member.The human growth hormone participates in many adjustings that normal human subject is grown.The molecular weight of about 22kDa formed and had by the naturally occurring strand pituitrin of this kind by 191 amino-acid residues.HGH represents numerous biological effects, especially comprise that (physique forms linear growth, the activation and the para-insulin effect of somatogenesis), lactation, scavenger cell and cause diabetes effect (Chawla, people such as R., Ann.Rev.Med.34:519-547 (1983); Isaksson, people such as O., Ann.Rev.Physiol, 47:483-499 (1985); Hughes, J. and Friesen, H., Ann.Rev.Physiol, 47:469-482 (1985)).
The structure of hGH is known (Goeddel, people such as D., Nature 281:544-548 (1979)) by us, and solves the three-dimensional structure (de Vos, people such as A., Science 255:306-312 (1992)) of hGH by X-ray crystallography.Described protein has compact ball-like structure, comprises four bundle of amphipathic alpha-helices, and it is called as the A-D by the ring connection from the beginning of N end.HGH also contains four cysteine residues, and it participates in forming two intramolecular disulfide bond: C53 and C165 pairing and C182 and C189 pairing.This hormone not by glycosylation and in intestinal bacteria (E.coli) with secretion type expression (Chang, people such as C, Gene 55:189-196 (1987)).
Many naturally occurring hGH mutant are differentiated.These mutant comprise hGH-V (Seeburg, DNA1:239 (1982); United States Patent (USP) the 4th, 446, No. 235, the 4th, 670, No. 393 and the 4th, 665, No. 180, it is incorporated herein by reference) and contain 20-kDa hGH (people such as Kostyo, the Biochem.Biophys.Acta 925:314 (1987) of disappearance of the residue 32-46 of hGH; Lewis, people such as U., J. Biol.Chem., 253:2679-2687 (1978)).In addition, reported by transcribing numerous hGH varients (Baumann, G., Endocrine Reviews 12:424 (1991)) that back, translation back, secretion, metabolism processing and other physiological process produce.
The biological effect of hGH is derived from the interaction of itself and specific cells acceptor.This hormone is the family member who comprises the homologous protein of placenta lactogen and prolactin.Yet, among the family member, hGH is uncommon, reason be it present specific specificity widely and with somatogenic (somatogenic) acceptor (Leung through the clone, D. wait the people, Nature330:537-543 (1987)) or prolactin acceptor (Boutin, people such as J., Cell 53:69-77 (1988)) combine.Based on structure and biochemical research, lactagogue has been proposed in conjunction with territory and somatogenic functional diagram (Cunningham, B. and Wells, J., Proc.Natl.Acad.Sci.88:3407 (1991)) in conjunction with the territory.The hGH acceptor is the member of hematopoiesis/cytokine/growth factor receptors family, described family comprises some other growth factor receptorses, such as, interleukin (IL)-3, interleukin-4 and Interleukin-6 acceptor, rHuGM-CSF (GM-CSF) acceptor, erythropoietin (EPO) acceptor and G-CSF acceptor.Referring to, Bazan, Proc.Natl.Acad.SciUSA 87:6934-6938 (1990).The member of cytokine receptor family is contained four conserved cysteine residue and tryptophane-Serine-X-tryptophane-Serine motif, and it just is positioned at strides outside the film district.Conserved sequence be considered to protein between interaction relevant, referring to, for example, people such as Chiba, Biochim.Biophys.Res.Comm.184:485-490 (1992).Interaction between hGH and its acceptor (hGHbp) extracellular domain is one of the best hormone-receptor interaction of understanding.High-resolution X-ray crystallization data (Cunningham, people such as B., Science, 254:821-825 (1991)) show that hGH has two receptor binding sites and uses the unique position on the molecule to combine with two acceptor molecules in order.Two receptor binding sites are called position I and position II.Position I comprises the C-terminal of spiral D and part and the A-B ring of spiral A, and position II is contained amino petiolarea and a part of spiral C of spiral A.GH and its acceptor combine with position I at first in conjunction with and take place in regular turn.Then, position II meshes the 2nd GH acceptor, causes receptor dimerization and the activation that causes signaling path in the cell of hormone generation cell response.G120R replace has been introduced among the II of position the hGH mutain can with single hGH receptors bind, but can not make two acceptor generation dimerizations.Mutain is estimated may be by occupying the receptor site but do not make signaling path activation in the cell and at the external hGH of serving as antagonist (Fuh, people such as G., Science 256:1677-1680 (1992)).
Reorganization hGH is used as therapeutical agent and has been given the ratification and is used for the treatment of many indications.The hGH shortage can cause (for example) nanism, and this illness has obtained the successful treatment of more than ten years by the exogenous dispensing of hormone.Except that hGH lacked, hGH also had been approved for treatment renal failure (in children), Turner's synodrome (Turner ' s Syndrome) and AIDS patient's emaciation.In the recent period, that hGH is used for the treatment of non-GH dependency is of short and small stature for Food and Drug Administration (FDA) approved.Aging, weakness, short bowel syndrome and the congestive heart failure that also hGH is being used for the treatment of the elderly at present investigated.The target colony of hGH treatment comprises children that suffer from the special property sent out (ISS) of short and small stature and the grownup with class GHD symptom.
At present, reorganization hGH sells as daily injectable product, and five kinds of main products: Humatrope are wherein arranged in the market TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono).Yet, use tethelin to be as the significant challenge of therapeutical agent, the protein transformation period in vivo is short, therefore and it must be thrown by the subcutaneous injection of every day and be obtained greatest treatment efficacy people such as (, J.Clin.Endocrinol.Metab.81:1806-1809 (1996)) MacGillivray.Make great efforts in a large number to concentrate on by reduce production costs, make with medicine throw give the patient easier, improve effect and security overview and produce other character that competitive advantage will be provided and improve on the method for dispensing of hGH agonist and antagonist.For example, Genentech and Alkermes sold Nutropin Depot in the past TM, the depot of a kind of hGH (depot) prescription, it is used for the paediatrics growth hormone deficiency.Though the depot prescription allows the dispensing (every 2-3 week once but not once a day) of lower frequency, it is also with descending such as bioavailability and the adverse side effect of place, injection site pain, and is withdrawn from market in 2004.In the recent period, another kind of product P egvisomant TM(Pfizer) also obtained the approval of FDA.Pegvisomant TMBe the genetically engineered analogue of hGH, it plays a part to be instructed to be used for the treatment of the highly selective growth hormone receptor antagonist (people such as van der Lely, The Lancet 358:1754-1759 (2001)) of acromegaly.Although Pegvisomant TMIn some amino acid side chain residues derived with polyoxyethylene glycol (PEG) polymkeric substance, but described product still will throw give once every day, this shows that pharmaceutics character is for best.Except that Pegylation and depot prescription, other dosing way that comprises hGH inhalant dosage form and oral dosage form be in clinical before and the commitment of clinical development, and all do not obtain the approval of FDA as yet.Therefore, to represent tethelin active but also provide longer serum half-life and (thereby) better the polypeptide of the treatment transformation period of hGH optimal treatment level and growth have demand.
The covalently bound many biologically active molecules that comprise protein, peptide for a kind of increase of poly-(ethylene glycol) (being abbreviated as PEG) of hydrophilic polymer, and especially be water-soluble, the biological usability of hydrophobic molecule, increase its serum half-life, increase its treatment transformation period, regulate its immunogenicity, the method for regulating its biological activity or prolonging its cycling time.PEG is widely used in the medicine, is used on artificial graft's thing, and be used for biocompatibility wherein, lack toxicity and lack immunogenicity using for very important other.PEG's wanted the character optimizing in order to make, the total molecular weight of the PEG polymkeric substance that is connected with bioactive molecules and hydration status must be enough high, with give usually the advantageous feature relevant with the connection of PEG polymkeric substance (such as, water-soluble and the circulating half-life that increases), can the biological activity of parent molecule not had a negative impact simultaneously.
The PEG derivative usually by reactive chemical functional group (such as, Methionin, halfcystine and histidine residues), N end and carbohydrate partly be connected in bioactive molecules.Protein and other molecule often have the reactive moieties that can be used for the limited quantity that polymkeric substance is connected.Usually, be adapted to pass through polymkeric substance most and connect the position of modifying and in receptors bind, bringing into play important effect, and be to keep the biological activity of molecule necessary.Thereby, polymer chain is connected to biological activity that these reactive moieties on the bioactive molecules usually can cause polymer-modified molecule indiscriminately significantly descends or even completely lose.People such as R.Clark, (1996), J.Biol.Chem, 271:21969-21977.Give the binding substances that target molecule is wanted enough polymericular weights of advantage for formation has, art methods is usually directed to being connected at random of numerous polymeric arms and molecule, and then has increased that the parent molecule biological activity reduces or even the danger that completely loses.
Being formed for making the reactive moieties of the locus that the PEG derivative is connected with protein is to arrange for protein structure.The protein that comprises enzyme is made up of various a-amino acid sequences, and described a-amino acid has general structure H 2N--CHR--COOH.The amino part of an amino acid whose α (H 2N--) with a contiguous amino acid whose carboxy moiety (--COOH) is connected with the formation amido linkage, described amido linkage can be expressed as--(NH--CHR--CO) n--, wherein subscript " n " may be for hundreds of or thousands of.The fragment of being represented by R can contain the reactive moieties that is useful on maintenance protein biological activity and is used to connect the PEG derivative.
For example, under the situation of amino acid lysine, on ε position and alpha position, have--NH 2Group.ε--NH 2But free responding under the alkaline pH condition.Make the many technology in the field of protein derivedization be directed to development in order to be connected to lysine residue ε--the NH that is present in the protein with PEG 2The PEG derivative of part." Polyethylene Glycol and Derivatives for Advanced PEGylation ", Nektar MolecularEngineering Catalog, 2003, the 1-17 pages or leaves.Yet these PEG derivatives all have common limitation, and promptly they can not optionally be positioned to be present in normal among the numerous lysine residue on the protein surface.Is under the very important situation at the lysine residue that for example is present in the zymophore to protein active, or under the situation that lysine residue works in the interaction of adjusting protein and other biomolecules, as under the situation of receptor binding site, this kind limitation can be significant limitations.
Second of existing protein PEGization method and complicacy of equal importance are that the PEG derivative can undesirable side reaction take place with the residue except those desirable residues.Histidine contains the reactive imino-part that is expressed as--N (H)--on the structure, but and ε--NH 2Many chemical reactivity materials of reaction also can react with--N (H)--.Similarly, the side chain of amino acid cysteine has on the structure and is expressed as-free sulfhydryl groups of SH.In some cases, be directed to the ε of Methionin--NH 2The PEG derivative also can with halfcystine, Histidine or the reaction of other residue.This can produce the complex heterogeneous mixture of PEG derivatize bioactive molecules, and emits the active danger that destruction institute target biology bioactive molecule is arranged.To wish development PEG derivative, it allows the chemical functional group is introduced single position in the protein, described chemical functional group then will make one or more PEG polymkeric substance can with bioactive molecules clear and definite and optionally coupling of predictable privileged site place on protein surface.
Except that lysine residue, a large amount of effort in this technology have been directed to the development of the activated PEG reagent of other amino acid side chain of target (comprising halfcystine, Histidine and N end).Referring to, for example, United States Patent (USP) the 6th, 610, No. 281, it is incorporated herein by reference; " Polyethylene Glycol and Derivatives for AdvancedPEGylation ", Nektar Molecular Engineering Catalog, 2003, the 1-17 pages or leaves.Can use known other technology in rite-directed mutagenesis and the affiliated field, cysteine residues is introduced in the proteinic structure in position selectivity mode, and gained free sulfhydryl groups part can with the PEG derivatives reaction with thiol-reactive functional group.Yet this method is complicated, and reason is that the introducing of free sulfhydryl groups can make gained protein expression, folding and stable complicated.Thereby, hope had a kind of the chemical functional group is introduced method in the bioactive molecules, its make one or more PEG polymkeric substance can with optionally coupling of protein, and simultaneously with sulfydryl and be present in usually other chemical functional group in the protein compatible (that is, not in undesirable side reaction with sulfydryl and other chemical functional group who is present in usually in the protein mesh).
As seen from the sampling in affiliated field, these be developed be used to connect proteinic side chain (be in particular and connect on the Methionin amino acid side chain--NH 2On part and the cysteine side chain-the SH part) derivative in many being proved to be at it in the synthetic and use arranged is problematic.Some derivatives and protein form unsettled key, described key be easy to take place hydrolysis and therefore decompose, degraded or not so in aqueous environment (such as, in blood flow) be unsettled.Some derivatives form more stable key, but before key forms hydrolysis take place easily, this mean reactive group on the PEG derivative may be before can connecting protein inactivation.Some derivatives have some toxicity and therefore are unsuitable in vivo using.To such an extent as to some derivatives reactions are too slow in fact inapplicable.Some derivatives cause the forfeiture of protein active by being connected with the position that produces protein active.Some derivatives are not specific to it with position of connecting, this also can cause the active forfeiture of wanting and cause lacking result's reproducibility.For overcoming and using the poly-relevant challenge of (ethylene glycol) part modifying protein, it is better (for example to have developed stability, United States Patent (USP) the 6th, 602, No. 498, it is incorporated herein by reference) or can with molecule and lip-deep thiol moiety generation selective reaction (for example, United States Patent (USP) the 6th, 610, No. 281, it is incorporated herein by reference) the PEG derivative.Obviously, need be in affiliated field for chemically inert in physiological environment up to requiring selective reaction to take place to form the PEG derivative till stablizing chemical bond.
In the recent period, reported a kind of brand new technical in the protein science field, it is hopeful to overcome with the protein site specific modifies relevant a lot of limitation.Specifically, new component has been added to the prokaryotic organism bacillus coli (Escherichia coli, E.coif) (for example, people such as L. Wang, (2001), Science292:498-500) with the eukaryote yeast saccharomyces cerevisiae (Sacchromyces cerevisiae, S.cerevisiae) (for example, people such as J.Chin, Science301:964-7 (2003)) in the protein biosynthesizing machine, described machine has made the amino acid of non-genetic coding can be in vivo incorporating in the protein.Use described method, with many amino acids with novel chemistry, physics or biological property (comprise photoaffinity labeling and photoisomerization amino acid, photocrosslinking amino acid (referring to, for example, Chin, people such as J.W., (2002) Proc.Natl.Acad.Sci.U.S.A.99:11020-11024; And Chin, people such as J.W., (2002) J.Am.Chem.Soc.124:9026-9027), ketone group amino acid, the amino acid that contains heavy atom and glycosylation amino acid) incorporate in the protein in intestinal bacteria and the yeast effectively and with high frequency high fidelity in response to amber codon TAG.Referring to, for example, people such as J.W.Chin, (2002), Journal of the American Chemical Society124:9026-9027; J.W.Chin and P.G. Schultz (2002), ChemBioChem3 (11): 1135-1137; People such as J.W.Chin, (2002), PNAS United States of America99:11020-11024; With L. Wang and P.G.Schultz, (2002), Chem.Comm., 1:1-11.All documents be to incorporate into by reference in full.Described research is verified, might be optionally and introduce as usual non-existent chemical functional group in the protein (such as, ketone group, alkynyl and nitrine part), described chemical functional group for all functional groups that exist in the amino acid of 20 kinds of common genetic codings for chemically inert and may be used for reacting effectively and optionally to form stable covalent linkage.
Incorporating the amino acid of non-genetic coding in the protein ability allow to introduce and can provide naturally occurring functional group (such as, the ε-NH of Methionin 2, sulfydryl-SH of halfcystine, the imino-of Histidine etc.) the chemical functional group of valuable surrogate.More known chemical functional groups for the functional group that exists in the amino acid of 20 kinds of common genetic codings for inert but can be fully and react effectively to form stable key.For example, Hu Shi root (Huisgen) [3+2] cycloaddition reaction is taking place in known azido-and ethynyl under aqueous conditions in the presence of the copper of catalytic amount in affiliated field.Referring to, for example, people such as Tornoe, (2002) J. Org.Chem.67:3057-3064; With people (2002) such as Rostovtsev Angew.Chem.Int.Ed.41:2596-2599.By partly introducing nitrine in the protein structure, for example, we can incorporate into for the amido that exists in the protein, sulfydryl, carboxylic acid group and hydroxyl for chemically inert but also can be successfully and effectively with the acetylene moiety reaction to form the functional group of cycloaddition product.Importantly under the situation that does not have acetylene moiety, the nitrine part still is chemically inert and anergy in the presence of other protein side chain and under physiology haemal strand spare.
The present invention especially is devoted to solve with the GH polypeptide active and makes a relevant difficult problem, and also is devoted to make and has the biology of improvement or pharmacological property the hGH polypeptide of (such as, improved treatment transformation period).
Summary of the invention
The invention provides and comprise one or more non-naturally encoded amino acid whose GH supergene family members, comprise GH (for example, hGH) polypeptide.
In certain embodiments, (for example, hGH) polypeptide comprises one or more posttranslational modifications to GH.In certain embodiments, (for example, hGH) polypeptide is connected with connexon, polymkeric substance or bioactive molecules GH.In certain embodiments, GH (for example, hGH) polypeptide and double functional copolymer, difunctionality connexon or GH that at least one is other (for example, hGH) polypeptide is connected.
In certain embodiments, non-naturally encoded amino acid is connected with water-soluble polymers.In certain embodiments, described water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, non-naturally encoded amino acid is to be connected with water-soluble polymers or to be connected with water-soluble polymers by key with connexon.In certain embodiments, poly-(ethylene glycol) molecule is the double functional copolymer.In certain embodiments, described double functional copolymer is connected with second polypeptide.In certain embodiments, described second polypeptide is GH (for example, a hGH) polypeptide.
In certain embodiments, (for example, hGH) polypeptide comprises at least two amino acid that are connected with the water-soluble polymers that comprises poly-(ethylene glycol) part to GH.In certain embodiments, at least one amino acid is non-naturally encoded amino acid.
(for example, each district hGH) can followingly illustrate GH, the amino acid position among the wherein middle line display hGH.
Spiral A spiral B spiral C spiral D
[1-5]-[6-33]-[34-74]-[75-96]-[97-105]-[106-129]-[130-153]-[154-183]-[184-191]
N end A-B ring B-C ring C-D ring C end
In certain embodiments, at following one or more any position in column regions: 1-5 (N end) down corresponding to the hGH secondary structure, 6-33 (spiral A), 34-74 (the zone between spiral A and the spiral B, the A-B ring), 75-96 (spiral B), 97-105 (the zone between spiral B and the spiral C, the B-C ring), 106-129 (spiral C), 130-153 (the zone between spiral C and the spiral D, the C-D ring), 154-183 (spiral D), 184-191 (C end) is (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3), the amino acid that one or more are non-naturally encoded is incorporated into.In other embodiments, in the position that is selected from the group that forms by following each residue position with non-naturally encoded aminoacid replacement: residue 1-5,32-46,97-105,132-149 and 184-191 (from hGH SEQID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the amino acid that one or more are non-naturally encoded (is for example incorporated GH into, hGH) one or more in are down in the column position: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).
In certain embodiments, in one or more following positions: position 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, in one or more following positions: position 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, in one or more following positions: position 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155 (SEQ IDNO:2, or the corresponding amino acid of SEQ ID NO:1 or 3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, in one or more following positions: position 30,74,103 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3), with one or more non-naturally encoded aminoacid replacement.In certain embodiments, in one or more following positions: position 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, the amino acid that the non-natural of one or more positions in including, but is not limited to these positions of following column position exists is connected with water-soluble polymers: position 1 is preceding (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the amino acid that the non-natural of one or more positions in described position exists is connected with water-soluble polymers: position 30,35,74,92,103,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the amino acid of the non-natural of one or more positions in described position existence is connected with water-soluble polymers: position 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).
Human GH antagonist includes, but is not limited in the position 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 and 127 places to have and replaces or 1 place has interpolation (promptly in the position, at N end) or its any combination (SEQ ID NO:2, or SEQ ID NO:1,3 or any other GH sequence in corresponding amino acid) those antagonists.
In certain embodiments, GH (for example, hGH) polypeptide comprise when replace, add with corresponding nothing or the GH of disappearance (regulation and control GH (for example when for example, avidity hGH) was compared, hGH) polypeptide is to GH (for example, hGH) replacement of the avidity of polypeptide receptor, interpolation or disappearance.In certain embodiments, (for example, hGH) polypeptide comprises when replacing, add with corresponding nothing or the GH of disappearance (makes GH (for example, the hGH) replacement, interpolation or the disappearance that increase of polypeptide stability when for example, stability hGH) being compared GH.In certain embodiments, (for example, hGH) polypeptide comprises the aminoacid replacement that is selected from the group that is made up of following each replacement to GH: F10A, F10H, F10I among the hGH SEQ ID NO:2; M14W, M14Q, M14G; H18D; H21N; G120A; R167N; D171S; E174S; F176Y, 1179T or its any combination.In certain embodiments, (for example, hGH) polypeptide comprises when replacing, add with corresponding nothing or the GH of disappearance (regulation and control GH when for example, immunogenicity hGH) is compared (for example, hGH) immunogenic replacement, interpolation or the disappearance of polypeptide GH.In certain embodiments, GH (for example, hGH) polypeptide comprises when replacing, add with corresponding nothing or the GH of disappearance (regulation and control GH when for example, serum half-life hGH) or cycling time compare (for example, hGH) replacement of the serum half-life of polypeptide or cycling time, interpolation or disappearance.
In certain embodiments, (for example, hGH) polypeptide comprises when replacing, add with corresponding nothing or the GH of disappearance (for example, making GH (for example, hGH) replacement of the water-soluble increase of polypeptide, interpolation or disappearance during hGH) water-soluble comparing GH.In certain embodiments, GH (for example, hGH) polypeptide comprises when replacing, add with corresponding nothing or the GH of disappearance (makes the GH that produces in the host cell (for example, the hGH) replacement, interpolation or the disappearance that increase of the solvability of polypeptide when for example, solvability hGH) being compared.In certain embodiments, GH (for example, hGH) polypeptide comprises when replacing, add with corresponding nothing or the GH of disappearance (for example, expression hGH) or syntheticly make GH in the host cell when comparing (for example, hGH) expression of polypeptides strengthens or makes in vitro synthetic enhanced replace, add or disappearance.In certain embodiments, the hGH polypeptide comprises aminoacid replacement G120A.The hGH polypeptide that comprises this replacement keeps the agonist activity and the expression level in the host cell is kept or raising.In certain embodiments, GH (for example, hGH) polypeptide comprise when replace, add with corresponding nothing or the GH of disappearance ((for example, hGH) the protease resistant enhanced of polypeptide replaces, adds or disappearance to make GH when for example, protease resistant hGH) is compared.
In certain embodiments, GH (for example, hGH) aminoacid replacement in the polypeptide can natural existence or the amino acid that exists of non-natural carry out, its restricted condition is to carry out with non-naturally encoded amino acid at least one replacement.
In certain embodiments, non-naturally encoded amino acid comprises carbonyl, ethanoyl, amino oxygen base, diazanyl, hydrazide group, amino urea groups, azido-or alkynyl.
In certain embodiments, non-naturally encoded amino acid comprises carbonyl.In certain embodiments, non-naturally encoded amino acid has following structure,
Figure A20058004446400161
Wherein n is 0-10, R 1Be alkyl, aryl, the alkyl that is substituted or the aryl that is substituted; R 2Be H, alkyl, aryl, the alkyl that is substituted and the aryl that is substituted; And R 3Be H, amino acid, polypeptide or aminoterminal modification group, and R 4Be H, amino acid, polypeptide or carboxyl terminal modification group.
In certain embodiments, non-naturally encoded amino acid comprises the amino oxygen base.In certain embodiments, non-naturally encoded amino acid comprises hydrazide group.In certain embodiments, non-naturally encoded amino acid comprises diazanyl.In certain embodiments, non-naturally encoded amino-acid residue comprises amino urea groups.
In certain embodiments, non-naturally encoded amino-acid residue comprises azido-.In certain embodiments, non-naturally encoded amino acid has following structure,
Figure A20058004446400162
Wherein n is 0-10; R 1For alkyl, aryl, the alkyl that is substituted, the aryl that is substituted or do not exist; X is O, N, S or does not exist; M is 0-10; R 2Be H, amino acid, polypeptide or aminoterminal modification group, and R 3Be H, amino acid, polypeptide or carboxyl terminal modification group.
In certain embodiments, non-naturally encoded amino acid comprises alkynyl.In certain embodiments, non-naturally encoded amino acid has following structure,
Figure A20058004446400171
Wherein n is 0-10; R 1Be alkyl, aryl, the alkyl that is substituted or the aryl that is substituted; X is O, N, S or does not exist; M is 0-10, R 2Be H, amino acid, polypeptide or aminoterminal modification group, and R 3Be H, amino acid, polypeptide or carboxyl terminal modification group.
In certain embodiments, polypeptide is GH (for example, hGH) a polypeptide agonist, part agonist, antagonist, partial antagonist or oppositely agonist.In certain embodiments, (for example, hGH) polypeptide agonist, part agonist, antagonist, partial antagonist or reverse agonist comprise the non-naturally encoded amino acid that is connected with water-soluble polymers to GH.In certain embodiments, water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, (for example, hGH) polypeptide agonist, part agonist, antagonist, partial antagonist or reverse agonist comprise non-naturally encoded amino acid and one or more posttranslational modifications, connexon, polymkeric substance or bioactive molecules to GH.In certain embodiments, the non-naturally encoded amino acid that is connected with water-soluble polymers is present in GH (for example, hGH) in the II zone, position of polypeptide (the protein zone that comprises amino terminal region and a part of spiral C of AC helical bundle surface, spiral A).In certain embodiments, (for example comprise the non-naturally encoded amino acid whose GH that is connected with water-soluble polymers, hGH) polypeptide is by preventing that GH (for example, hGH) polypeptide antagonist and second GH are (for example, hGH) the polypeptide receptor molecule combines and prevents GH (for example, hGH) polypeptide receptor generation dimerization.In certain embodiments, with the G120 among the aminoacid replacement SEQ ID NO:2 (hGH) of non-glycine.In certain embodiments, with the G120 among the arginine replacement SEQ IDNO:2.In certain embodiments, with the G120 among the non-naturally encoded aminoacid replacement SEQ ID NO:2.In certain embodiments, the non-naturally encoded amino acid that is connected with water-soluble polymers is present in GH (for example, hGH) in the receptor binding domain of polypeptide or disturb GH (for example, the hGH) receptors bind of polypeptide.
The present invention also provides separated nucleic acid, and it is included under the stringent condition polynucleotides with SEQ ID NO:21 or 22 hybridization, and wherein said polynucleotide comprises at least one and selects codon.In certain embodiments, described selection codon is to be selected from by the molecular group of following password: amber codon, ochre (ochre) codon, opal codon, single password, rare codon, five base codons and four base codons.
GH (for example, the hGH) method of polypeptide that the present invention also provides preparation to be connected with water-soluble polymers.In certain embodiments, described method comprise make comprise non-naturally encoded amino acid whose separated GH (for example, hGH) polypeptide with comprise and can contact with the water-soluble polymers of the part of described non-naturally encoded amino acid reaction.In certain embodiments, (for example, hGH) the non-naturally encoded amino acid in the polypeptide has reactivity for water-soluble polymers, and described water-soluble polymers does not but have reactivity in 20 kinds of common amino acids any in addition to incorporate GH into.In certain embodiments, (for example incorporate GH into, hGH) the non-naturally encoded amino acid in the polypeptide has reactivity for connexon, polymkeric substance or bioactive molecules, and described connexon, polymkeric substance or bioactive molecules but do not have reactivity in 20 kinds of common amino acids any in addition.
In certain embodiments, by make comprise contain carbonyl amino acid whose GH (for example, hGH) polypeptide and poly-(ethylene glycol) molecular reaction that comprises amino oxygen base, diazanyl, hydrazide group or amino urea groups make the GH that is connected with water-soluble polymers (for example, hGH) polypeptide.In certain embodiments, by amido linkage amino oxygen base, diazanyl, hydrazide group or amino urea groups are connected with poly-(ethylene glycol) molecule.
In certain embodiments, by making poly-(ethylene glycol) molecule of comprising carbonyl and comprising the non-naturally encoded amino acid whose polypeptide that contains amino oxygen base, diazanyl, hydrazide group or amino urea groups and react and make the GH that is connected with water-soluble polymers (for example, hGH) polypeptide.
In certain embodiments, comprise the amino acid whose GH that contains alkynyl (for example, hGH) polypeptide and poly-(ethylene glycol) molecular reaction that comprises the nitrine part make the GH that is connected with water-soluble polymers (for example, hGH) polypeptide by making.In certain embodiments, by amido linkage azido-or alkynyl are connected with poly-(ethylene glycol) molecule.
In certain embodiments, comprise the amino acid whose GH that contains azido-(for example, hGH) polypeptide and poly-(ethylene glycol) molecular reaction that comprises alkynyl moiety make the GH that is connected with water-soluble polymers (for example, hGH) polypeptide by making.In certain embodiments, by amido linkage azido-or alkynyl are connected with poly-(ethylene glycol) molecule.
In certain embodiments, poly-(ethylene glycol) molecule has the molecular weight between about 0.1kDa and about 100kDa.In certain embodiments, poly-(ethylene glycol) molecule has the molecular weight between 0.1kDa and 50kDa.
In certain embodiments, poly-(ethylene glycol) molecule is a branched polymers.In certain embodiments, each of poly-(ethylene glycol) branched polymers has between 1kDa and the 100kDa or the molecular weight between 1kDa and 50kDa.
In certain embodiments, (for example, hGH) water-soluble polymers that is connected of polypeptide comprises polyalkylene glycol moiety with GH.In certain embodiments, (for example, hGH) the non-naturally encoded amino-acid residue in the polypeptide comprises carbonyl, amino oxygen base, hydrazide group, diazanyl, amino urea groups, azido-or alkynyl to incorporate GH into.In certain embodiments, (for example, hGH) the non-naturally encoded amino-acid residue in the polypeptide comprises carbonyl moiety and water-soluble polymers comprises amino oxygen base, hydrazides, hydrazine or Urea,amino-part to incorporate GH into.In certain embodiments, (for example, hGH) the non-naturally encoded amino-acid residue in the polypeptide comprises alkynyl moiety and water-soluble polymers comprises the nitrine part to incorporate GH into.In certain embodiments, (for example, hGH) the non-naturally encoded amino-acid residue in the polypeptide comprises the nitrine part and water-soluble polymers comprises alkynyl moiety to incorporate GH into.
The present invention also provides to comprise and contains non-naturally encoded amino acid whose GH (for example, the hGH) composition of polypeptide and pharmaceutically acceptable supporting agent.In certain embodiments, non-naturally encoded amino acid is connected with water-soluble polymers.
The present invention also provide comprise the coding GH (for example, the hGH) cell of the polynucleotide of polypeptide, described polynucleotide comprises the selection codon.In certain embodiments, described cell comprises and is used for non-naturally encoded aminoacid replacement is gone into GH (for example, hGH) quadrature RNA synthetic enzyme and/or the quadrature tRNA in the polypeptide.
The present invention also provides preparation to comprise non-naturally encoded amino acid whose GH (for example, the hGH) method of polypeptide.In certain embodiments, described method comprise under certain condition cultivate comprise coding GH (for example, hGH) cell of the polynucleotide of polypeptide, quadrature RNA synthetic enzyme and/or quadrature tRNA to allow GH (for example, hGH) expression of polypeptides; With purifying from the GH of described cell and/or substratum (for example, hGH) polypeptide.
The present invention also provides GH (for example, the method that hGH) prolongs the treatment transformation period of polypeptide, serum half-life or cycling time that makes.The present invention also provides regulation and control GH (for example, hGH) the immunogenic method of polypeptide.In certain embodiments, described method with the naturally occurring GH of non-naturally encoded aminoacid replacement (for example comprises, hGH) any one in the polypeptide or one are with upper amino acid and/or make described GH (for example, hGH) polypeptide is connected with connexon, polymkeric substance, water-soluble polymers or bioactive molecules.
The present invention provides also that (for example, hGH) molecular therapy needs the patient's of this treatment method with the GH of the present invention of significant quantity.In certain embodiments, described method comprises the GH that comprises with the treatment significant quantity (for example, hGH) medical composition of polypeptide and pharmaceutically acceptable supporting agent is thrown and given described patient, and (for example, hGH) polypeptide comprises non-naturally encoded amino acid to described GH.In certain embodiments, non-naturally encoded amino acid is connected with water-soluble polymers.
The present invention also provide comprise the sequence shown in the SEQ ID NO:1,2,3 or any other GH peptide sequence (except that at least one amino acid by the non-naturally encoded aminoacid replacement) GH (for example, hGH) polypeptide.In certain embodiments, described non-naturally encoded amino acid is connected with water-soluble polymers.In certain embodiments, described water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, non-naturally encoded amino acid comprises carbonyl, amino oxygen base, hydrazide group, diazanyl, amino urea groups, azido-or alkynyl.In certain embodiments, in the position that is selected from the group that forms by following each residue position with non-naturally encoded aminoacid replacement: from residue 1-5,82-90,117-134 and the 169-176 of SEQ ID NO:3 (hGH).
The present invention also provides and (for example comprises pharmaceutically acceptable supporting agent and GH, hGH) medical composition of polypeptide, described GH (for example, hGH) polypeptide comprises the sequence shown in the SEQ ID NO:1,2,3 or any other GH peptide sequence, and wherein at least one amino acid is by non-naturally encoded aminoacid replacement.In certain embodiments, described non-naturally encoded amino acid comprises the carbohydrate part.In certain embodiments, by carbohydrate water-soluble polymers is connected with described polypeptide.In certain embodiments, (for example, hGH) polypeptide is connected partly to make connexon, polymkeric substance or bioactive molecules and GH by carbohydrate.
The present invention also provides and comprises (for example, the hGH) GH of the water-soluble polymers that is connected at the single amino acids place of polypeptide (for example, the hGH) polypeptide by covalent linkage and GH.In certain embodiments, water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, the amino acid that covalently is connected with water-soluble polymers is the non-naturally encoded amino acid that is present in the described polypeptide.In certain embodiments, in the position 35,92,143 of SEQ ID NO 2 or 145 places with non-naturally encoded aminoacid replacement.
The invention provides comprise at least one connexon, polymkeric substance or bioactive molecules GH (for example, hGH) polypeptide wherein makes described connexon, polymkeric substance or bioactive molecules be connected with described polypeptide by the non-naturally encoded amino acid whose functional group that incorporates into the rrna effect in the described polypeptide.In certain embodiments, described polypeptide coverlet PEGization.The present invention also provide comprise the connexon, polymkeric substance or the bioactive molecules that are connected with one or more non-naturally encoded amino acid GH (for example, hGH) polypeptide wherein acts on previously selected position with rrna described non-naturally encoded amino acid is incorporated in the described polypeptide.
In another embodiment, comprise the amino acid whose hGH polypeptide that one or more non-naturals exist and provide pure substantially hGH with the combining of molecule that another includes, but is not limited to PEG owing to combining the unique chemical reaction that is utilized with described alpha-non-natural amino acid.Comprising one or more non-naturally encoded amino acid whose hGH can carry out so that pure substantially hGH to be provided with other purification technique of carrying out before or after integrating step with another combination such as the molecule of PEG.
The present invention further provides a kind of hormonal composition that contains the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, wherein said covalent linkage is the oxime key.In certain embodiments, described GH is such as the human growth hormone (hGH) at least about 80% sequence identical with SEQ ID NO:2; In certain embodiments, described sequence is the sequence of SEQ ID NO:2.Described GH can comprise one or more non-naturally encoded amino acid (NEAA), and () NEAA for example, ketone group is such as being NEAA to acetylphenylalanine such as comprising carbonyl.In certain embodiments, described oxime key is between NEAA and water-soluble polymers.Described GH can corresponding to the position of the position 35 of SEQID NO:2 through acetylphenylalanine is replaced.In certain embodiments, described water-soluble polymers comprises one or more polyoxyethylene glycol (PEG) molecule.Described PEG can be linear, and for example, molecular weight is between about 0.1kDa and the about 100kDa or between about 1kDa and the about 60kDa or between about 20kDa and about 40kDa or be the linear PEG of about 30kDa.In certain embodiments, described PEG is branch PEG, and for example, molecular weight is between about 1kDa and the about 100kDa or between about 30kDa and about 50kDa or be the branch PEG of about 40kDa.In certain embodiments, by a plurality of covalent linkage GH is connected with a plurality of water-soluble polymerss, wherein at least one covalent linkage is the oxime key.Among some embodiment in these embodiments, described GH be the human growth hormone (GH, for example, hGH), for example sequence has at least about 80% GH identical with SEQ ID NO:2 (for example, hGH); Described in certain embodiments sequence is the sequence of SEQ ID NO:2.GH (for example, hGH) with some embodiment that a plurality of water-soluble polymerss are connected in, described GH comprises a plurality of NEAA.
In certain embodiments, the invention provides a kind of contain comprise SEQ ID NO:2 sequence GH (for example, hGH) GH composition, wherein make GH (for example by the oxime key, hGH) be connected with the linear PEG of 30kDa, and wherein said oxime key is and replace in the position corresponding to the position 35 of SEQ ID NO:2 acetylphenylalanine is formed.
In certain embodiments, the invention provides and a kind ofly (for example contain the GH that is connected by the linear PEG of oxime key and at least one, hGH) hormonal composition, wherein said GH (for example, hGH) comprises the aminoacid sequence of SEQ ID NO:2 and contain the NEAA that at least one replaces in one or more positions that are selected from the group that is made up of following each residue position: residue 1-5,6-33,34-74,75-96,97-105,106-129,130-153,154-183 and 184-191.In certain embodiments, replace with NEAA in one or more positions that are selected from the group that forms by following each residue position: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191 and 192 (that is, at proteinic carboxyl terminales).In certain embodiments, replace with NEAA in one or more positions that are selected from the group that forms by residue position 35,92,131,134,143 and 145.In certain embodiments, replace with NEAA in one or more positions that are selected from the group that forms by residue position 30,35,74,92,103,143 and 145.In certain embodiments, replace with NEAA in one or more positions that are selected from the group that forms by residue position 35,92,143 and 145.In certain embodiments, 35 places replace with NEAA in the position.In certain embodiments, at least one NEAA is to acetylphenylalanine.In certain embodiments, the molecular weight of PEG or between about 1kDa and about 60kDa, or between about 20kDa and about 40kDa, or is about 30kDa between about 0.1kDa and about 100kDa.
In other embodiments, (for example the invention provides GH that a kind of preparation is connected with water-soluble polymers by the oxime key, hGH) method, it comprises (for example, hGH) contacts the GH that comprises the NEAA that contains carbonyl being suitable for forming under the condition of oxime key with the PEG oxyamine.Described NEAA can contain ketone group, for example, and carbonyl.NEAA can be acetylphenylalanine.In some embodiment that contain acetylphenylalanine, (for example, acetylphenylalanine is replaced with described corresponding to the position of the amino acid 35 among the SEQ ID NO:2 in hGH) at GH.In certain embodiments, the PEG oxyamine is mono methoxy PEG (MPEG) oxyamine.In certain embodiments, the MPEG oxyamine is linear, the linear MPEG of for example about 20-40kDa or about 30kDa.In certain embodiments, the MPEG oxyamine is linear 30kDa mono methoxy-PEG-2-amino oxygen base ethamine carbamate hydrochloride.In certain embodiments, by (i) (for example encoded GH, hGH) nucleic acid (wherein said nucleic acid has been modified so that select codon for NEAA incorporates into to provide) and (ii) NEAA introduce make in the organism comprise NEAA GH (for example, hGH), described organic cell machine can be incorporated NEAA in the protein in response to the selection codon of (i) nucleic acid.In certain embodiments, the reaction conditions that forms the oxime key comprise with MPEG and GH (for example, hGH) mix with generation MPEG-GH (for example, hGH) mixture, wherein (for example, ratio hGH) is about 5 to 10 to MPEG:GH, and pH is about 4 to 6; At room temperature with described MPEG-GH (for example, MPEG-hGH) the soft stir about of mixture 10 to 50 hours.In certain embodiments, described method comprises that further (for example, hGH) purifying (for example) to purity is at least about 99% with GH.
The cell machine includes, but is not limited to quadrature tRNA and/or aminoacyl tRNA synthetase.
In other embodiments, the invention provides a kind of medical composition that contains hormonal composition and pharmaceutically acceptable vehicle, described hormonal composition comprises the tethelin that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.In certain embodiments, GH for example is the GH of hGH.In certain embodiments, GH comprises NEAA.In certain embodiments, water-soluble polymers comprises PEG, such as linear PEG.In certain embodiments, PEG is the linear PEG of about 30kDa, and GH be corresponding to the position of the amino acid 35 of SEQ ID NO:2 through to the GH of acetylphenylalanine replacement (for example, hGH), and between to acetylphenylalanine and PEG formation oxime key.
In certain embodiments, the invention provides a kind of method for the treatment of by the individuality that the hormonal composition throwing of significant quantity is given the needs treatment, described hormonal composition contains the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.In certain embodiments, GH is hGH.In certain embodiments, GH comprises NEAA.In certain embodiments, water-soluble polymers comprises PEG, such as linear PEG.In certain embodiments, PEG is the linear PEG of about 30kDa, and GH be corresponding to the position of the amino acid 35 of SEQ ID NO:2 through hGH to the acetylphenylalanine replacement, and between to acetylphenylalanine and PEG formation oxime key.In certain embodiments, the individuality of being treated is for human.In certain embodiments, the individuality of being treated suffer from the paediatrics growth hormone deficiency, special send out property of short and small stature, the Childhood outbreak grownup's growth hormone deficiency, the grownup's growth hormone deficiency or the Secondary cases growth hormone deficiency of Adulthood outbreak.In certain embodiments, GH is hGH.In certain embodiments, GH comprises NEAA.In certain embodiments, water-soluble polymers comprises PEG, such as linear PEG.In certain embodiments, PEG is the linear PEG of about 30kDa, and GH (for example, hGH) be corresponding to the position of the amino acid 35 of SEQID NO:2 through acetylphenylalanine is replaced, and between to acetylphenylalanine and PEG formation oxime key.
In certain embodiments, the invention provides a kind of method for the treatment of by the individuality that the hormonal composition throwing of significant quantity is given the needs treatment, described hormonal composition comprises the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, wherein said water-soluble polymers is a linear polymer, and wherein with weekly only once, whenever biweekly or mensal frequency throw and to give described hormonal composition.In certain embodiments, described polymkeric substance is PEG.In certain embodiments, described GH comprises NEAA.In certain embodiments, by the oxime key described polymkeric substance is connected with described GH.
In certain embodiments, the invention provides a kind of GH of comprising (for example, hormonal composition hGH), wherein (for example, average serum transformation period hGH) is at least about 12 hours as described GH when Mammals is given in subcutaneous throwing.In certain embodiments, GH (for example, hGH) comprises NEAA.In certain embodiments, described hormonal composition further contains water-soluble polymers, such as, PEG, for example, linear PEG.
In certain embodiments, the invention provides and a kind ofly (for example comprise the GH that is connected with PEG, hGH) hormonal composition, wherein when described GH when Mammals is given in subcutaneous throwing (for example, hGH) the average serum transformation period is the GH that comprises no PEG (for example, the serum half-life of composition hGH) at least about 7 times.
Description of drawings
Description of drawings
Fig. 1 shows the proteic general structure iron of four-helix bundle.
Fig. 2 shows the general structure iron of four-helix bundle albumen tethelin (GH).
Fig. 3 shows the general structure iron of four-helix bundle albumen erythropoietin (EPO).
Fig. 4 shows the general structure iron of four-helix bundle albumen interferon (IFN α-2).
Fig. 5 shows the general structure iron of four-helix bundle protein granules colony-stimulating factor (G-CSF).
Fig. 6 shows coomassie (Coomassie) light blue dyeing SDS-PAGE, and it is illustrated in each position in the upper/lower positions and comprises the expression of non-naturally encoded amino acid to the hGH of acetylphenylalanine: Y35, F92, Y111, G131, R134, K140, Y143 or K145.
Fig. 7, A figure and Fig. 7, B figure demonstration comprises non-naturally encoded amino acid whose hGH (B figure) and the bioactive figure of wild-type hGH (A figure) in the IM9 cell.
Fig. 8 shows the coomassie brilliant blue staining SDS-PAGE that shows the generation comprise non-naturally encoded amino acid whose hGH, described hGH be by PEG (5kDa, 20kDa and 30kDa) with the covalently bound of its PEGization.
Fig. 9 shows that displaying comprises the bioactive figure of multiple PEGization form in the IM9 cell of non-naturally encoded amino acid whose hGH.
Figure 10, A figure-this figure illustrates the primary structure of hGH, wherein indicates trypsinase cracking position and with arrow non-natural aminoacid replacement F92pAF (revising the figure of gained according to people Biotechnol Appl Biochem. (1988) 10 (4): 326-337 such as Becker) is described.Figure 10, B figure-demonstration by comprise peptide (being labeled as A) that non-naturally encoded amino acid whose PEGization hGH polypeptide produces, by the synergetic trypsinase figure of the peptide (being labeled as C) that comprises peptide (being labeled as B) that non-naturally encoded amino acid whose hGH polypeptide produces and produce by WHO rhGH.Figure 10, figure C-shows the amplification from the peak 9 of B figure.
Figure 11, A figure and B figure show that the coomassie brilliant blue staining SDS-PAGE of purified PEG-hGH polypeptide analyzes.
Figure 12 shows the bioactive figure of hGH dimer molecule in the IM9 cell.
Figure 13, the figure that makes the IM-9 calibrating data of pSTAT5 phosphorylation by the hGH antagonist with G120R replacement is measured in A figure-demonstration.Figure 13, the figure that makes the IM-9 calibrating data of pSTAT5 phosphorylation by the hGH polypeptide with alpha-non-natural amino acid of incorporating at same position place (G120) is measured in B figure-demonstration.
Figure 14 demonstration shows Figure 13, and the dimer of the hGH antagonist shown in the B figure also lacks bioactive figure in the IM-9 calibrating.
Figure 15 shows and relatively to comprise the non-naturally encoded amino acid whose PEGization hGH polypeptide and the figure of the serum half-life of hGH polypeptide in rat of PEGization not.
Figure 16 shows the figure that relatively comprises the non-naturally encoded serum half-life of amino acid whose PEGization hGH polypeptide in rat.
Figure 17 shows the figure that relatively comprises the non-naturally encoded serum half-life of amino acid whose PEGization hGH polypeptide in rat.Once throw and give 2.1mg/kg to rat.
Figure 18, A scheme-are presented to throw behind the PEGization hGH polypeptide give comprising of single dose of non-naturally encoded amino acid (position 35,92) figure to the influence of rat body weight increase.Figure 18, B scheme-are presented to throw behind the PEGization hGH polypeptide give comprising of single dose of non-naturally encoded amino acid (position 35,92) figure to the influence of circulating plasma IGF-1 level.Figure 18, C scheme-are presented to throw behind the PEGization hGH polypeptide give comprising of single dose of non-naturally encoded amino acid (position 92,134,145,131,143) figure to the influence of rat body weight increase.Figure 18, D scheme-are presented to throw behind the PEGization hGH polypeptide give comprising of single dose of non-naturally encoded amino acid (position 92,134,145,131,143) figure to the influence of circulating plasma IGF-1 level.Figure 18, E figure-demonstration relatively comprises the figure of the serum half-life of PEGization hGH polypeptide in rat of non-naturally encoded amino acid (position 92,134,145,131,143).
Figure 19 shows the structure iron of mono methoxy-poly-(ethylene glycol)-2-amino oxygen base ethamine carbamate hydrochloride of straight chain, 30kDa.
Figure 20 explicit declaration is through the synthetic figure of the oxygen base amino derivatization PEG of carbamate connection.
Figure 21 present can be used to modify non-natural amino acid polypeptides with formation contain PEG, the illustrative that contains PEG reagent, the limiting examples of the non-natural amino acid polypeptides that connects through oxime.
Figure 22 present synthetic can be used to modify non-natural amino acid polypeptides with formation contain PEG, the illustrative that contains PEG reagent, the limiting examples of the non-natural amino acid polypeptides that connects through oxime.
Figure 23 present synthetic can be used to modify non-natural amino acid polypeptides with formation contain PEG, the illustrative that contains azanol PEG reagent, the limiting examples based on acid amides of the non-natural amino acid polypeptides that connects through oxime.
Figure 24 present synthetic can be used to modify non-natural amino acid polypeptides with formation contain PEG, the illustrative that contains PEG reagent, the limiting examples based on carbamate of the non-natural amino acid polypeptides that connects through oxime.
Figure 25 present synthetic can be used to modify non-natural amino acid polypeptides with formation contain PEG, the illustrative that contains PEG reagent, the limiting examples based on carbamate of the non-natural amino acid polypeptides that connects through oxime.
Figure 26 present synthetic can be used to modify non-natural amino acid polypeptides with formation contain PEG, the illustrative that simply contains PEG reagent, the limiting examples of the non-natural amino acid polypeptides that connects through oxime.
Figure 27 present can be used to modify non-natural amino acid polypeptides with formation contain PEG, the illustrative that contains branch PEG reagent, limiting examples and a kind of described reagent of the non-natural amino acid polypeptides that connects through oxime modifies the purposes based on the non-natural amino acid polypeptides of carbonyl.
Figure 28 presents the PEG-that explanation increases with placebo or dosage weekly aHGH or every day are with the figure of the IGF-1 plasma concentration in the rat that hypophysectomizes of placebo or strong person of outstanding talent peaceful (Genotropin) treatment.
Figure 29 presents explanation weekly with placebo or PEG- aHGH or every day are with the figure of the shin bone length in the rat that hypophysectomizes of placebo or the peaceful treatment of strong person of outstanding talent.
Figure 30 presents explanation weekly with placebo or PEG- aHGH or every day are with the figure of the body weight change per-cent in the rat that hypophysectomizes of placebo or the peaceful treatment of strong person of outstanding talent.
The PEG-that Figure 31 presents explanation the 0th day and gave through subcutaneous throwing in the 7th day aThe plasma concentration of hGH and the figure of time relation.
Figure 32 presents explanation and throws the PEG-that gives with subcutaneous or intravenously single dose a(met) figure of the plasma concentration of hGH and time relation.
Embodiment
Definition
Should be appreciated that the present invention is not subject to ad hoc approach described herein, experimental program, clone, construction and reagent, and thereby may change.Should be appreciated that also term used herein only is in order to describe specific embodiment, rather than is used for limiting category of the present invention, category of the present invention will only be subject to the claims of enclosing.
Unless context offers some clarification in addition, otherwise as herein and enclose in claims employed singulative " " and " as described in " comprise plural implication.Therefore, for example, mention that (kind) " hGH " mentions one (kind) or protein and comprise known its equivalent of those skilled in the art so more than one (kind), or the like.
Except as otherwise noted, otherwise all scientific and technical terminologies used herein have the implication identical with those skilled in the art in the invention's common sense.Though can use and those similar or equivalent any method, device and materials described herein in practice of the present invention or test, what describe now is preferred method, device and material.
All open cases mentioned herein and patent are to be incorporated herein by reference, and describe and disclose the construction that (for example) described, may be used in combination with the invention of current description and the purpose of method to reach in described open case.The open case of discussing herein that is provided only is its disclosure before the date of application of the application's case.This paper where face in office is neither should to be interpreted as admitting that date that the present inventor haves no right to make described disclosure owing to existing invention or any other reason in advance.
Term " purifying substantially " (for example refers to following GH, hGH) polypeptide, at the GH that produces with recombination method (for example, hGH) under the situation of polypeptide, it can substantially or be substantially devoid of usually with as seen in the protein in its naturally occurring environment (that is, n cell or host cell) or with described protein interactional component taking place.Can not contain substantially cellular material GH (for example, hGH) polypeptide comprise contaminating protein matter be lower than about 30%, be lower than about 25%, be lower than about 20%, be lower than about 15%, be lower than about 10%, be lower than about 5%, be lower than about 4%, be lower than about 3%, be lower than about 2% or be lower than the protein formulation of about 1% (with dry weight basis).When GH (for example, hGH) polypeptide or its varient are when being produced by host cell by recombination method, protein can dry cell weight about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% about 1% or the amount of lower per-cent exist.When GH (for example, hGH) polypeptide or its varient are when being produced by host cell by recombination method, and protein can about 5g/L, about 4g/L, the amount of about 3g/L, about 2g/L, about 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 lower dry cell weight is present in the substratum.Thereby, GH (for example as " purifying substantially " that produced by method of the present invention, hGH) polypeptide can have at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, purity at least about 70%, especially for having at least about 75%, 80%, 85% purity, and more particularly for having purity at least about 90%, purity at least about 95%, at least about 99% or the purity of bigger value, described purity is by analyzing such as SDS/PAGE, RP-HPLC, the proper method of SEC and capillary electrophoresis is measured.
" recombinant host cell " or " host cell " refers to the cell of the exogenous polynucleotide that comprises which kind of method that don't work (other method of known generation recombinant host cell in for example, directly absorption, transduction, f joint or this technology) is inserted.Described exogenous polynucleotide can be used as the nonconformity carrier, and for example plasmid is maintained, and perhaps, can be incorporated in the host genome.
As used herein term " substratum " comprises any substratum, solution, solid, semisolid or rigid support thing, it can support or contain any host cell and cell content, and described host cell comprises 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, intestinal bacteria or pseudomonas (Pseudomonas) host cell.Thereby described term can be contained host cell and grow in wherein substratum, and for example, (for example, hGH) polypeptide has been secreted into substratum wherein to GH, comprises the substratum before or after the multiplicative stage.Described term also can contain such as GH (for example, hGH) polypeptide be produce in the cell and make host cell dissolving or division (for example, hGH) under the situation of polypeptide, contain the damping fluid or the reagent of the molten born of the same parents' thing of host cell to discharge GH.
As being to be defined as to make sulfydryl maintain reduced state and make intramolecularly or any compound or the material of intermolecular disulfide bond reduction about protein refolding employed " reductive agent " herein.Appropriate reductant includes, but is not limited to dithiothreitol (DTT) (DTT), 2 mercapto ethanol, dithioerythritol, halfcystine, cysteamine (2-aminoothyl mercaptan) and reduced glutathion.The those skilled in the art is easy to understand, and multiple reductive agent is applicable in the method and composition of the present invention.
As being to be defined as any compound or the material that to remove electronics from just oxidized compound about protein refolding employed " oxygenant " herein.Suitable oxygenant comprises (but being not limited to) Sleep-promoting factor B, Gelucystine, cystamine, oxidized form dithiothreitol (DTT), the red threitol of oxidized form (oxidized erythreitol) and oxygen.The those skilled in the art is easy to understand, and multiple oxygenant is applicable in the method for the present invention.
" denaturing agent " is to be defined as to cause that protein generation reversible separates folding any compound or material as used herein.The intensity of denaturing agent will be determined by the character and the concentration of specific denaturing agent.Suitable denaturing agent can be chaotropic agent, stain remover, organic solvent, water miscible solvent, phosphatide or two or more described combination of agents.Suitable chaotropic agent includes, but is not limited to urea, guanidine and Sodium Thiocyanate 99.The available stain remover can include, but is not limited to the strong dirt-removing agent (such as, sodium lauryl sulphate or Soxylat A 25-7 are (for example, tween (Tween) or Triton (Triton) stain remover), N-sarcosyl (sarkosyl)), gentle non-ionic detergent (for example, digitonin), gentle catioic detergent (such as, N-〉2,3-(two oily acyloxy)-propyl group-N, N, the N-TMA (TriMethylAmine)), gentle ionic detergent (for example, Sodium cholic acid or Sodium desoxycholate) or the amphoteric ion type stain remover (include, but is not limited to sultaine (Zwittergent, ampholytic detergent), 3-(3-courage amido propyl) dimethylin-1-propane vitriol (CHAPS) and 3-(3-courage amido propyl) dimethylin-2-hydroxyl-1-propane sulfonate (CHAPSO)).(be in particular C such as acetonitrile, low-carbon (LC) alkanol 2-4Alkanol, such as, ethanol or Virahol) or the lower alkanes glycol (be in particular C 2-4The alkane glycol is such as, ethylene glycol) organic water miscible solvent can be used as denaturing agent.Can be used for phosphatide of the present invention can be naturally occurring phosphatide (such as; phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositols) or synthetic phospholipid derivative or variant (such as, two caproyl phosphatidylcholines or two oenanthyl phosphatidylcholines).
As used herein " refolding " describe and with regard to disulfide linkage, to make the polypeptide that contains disulfide linkage from inappropriate folding or separate any process, reaction or the method that folded state changes natural or appropriate folded conformation into.
" altogether folding " refers specifically at least two interactional to each other polypeptide of employing and makes refolding process, reaction or the method that folding or incorrect folding polypeptide changes natural, appropriate folding polypeptide into of separating as used herein.
" tethelin " or " GH " as used herein, no matter how and in addition its biological activity no matter which kind of synthetic or manufacture method it adopts (include, but is not limited in vitro, in vivo recombinate (no matter from cDNA by the microinjection of nucleic acid molecule, genomic dna, the nucleic acid that synthetic DNA produces still from other form produces) method, synthetic method, transgenic method and gene activation method), it all should comprise at least a bioactive those polypeptides and the protein with tethelin, described tethelin (includes, but is not limited to people (hGH) from any mammal, ox (bGH), and comprise its GH analogue pig) and other livestock or domestic animal (including, but is not limited to chicken),, GH is with the merit iso series, the GH stand-in, the GH fragment, hybridization GH albumen, fusion rotein, oligomer and polymer, homologue, glycosylation form variation body, varient, splicing variants and mutein.
The hGH polypeptide that comprises one or more aminoacid replacement, interpolation or disappearance contained in term " hGH polypeptide ".Exemplary replacement comprises the replacement of position 41 place's Methionins of (for example) natural hGH or the replacement of position 176 place's phenylalanines.In some cases, be 41 places in the position if replace, substituent can be Isoleucine or arginine residues so, if or the position be 176, substituent is a tyrosine residues so.Position F10 can replace through (for example) A, H or I.Position M14 can replace through (for example) W, Q or G.Other exemplary replacement comprises any including (but not limited to) following each replacement that replaces or its combination: R167N, D171S, E174S, F176Y, I179T; R167E, D171S, E174S, F176Y; F10A, M14W, H18D, H21N; F10A, M14W, H18D, H21N, R167N, D171S, E174S, F176Y, I179T; F10A, M14W, H18D, H21N, R167N, D171A, E174S, F176Y, I179T; F10H, M14G, H18N, H21N; F10A, M14W, H18D, H21N, R167N, D171A, T175T, I179T; Or F10I, M14Q, H18E, R167N, D171S, I179T.Referring to, for example, United States Patent (USP) the 6th, 143, No. 523, it is incorporated herein by reference.
The exemplary replacement on a plurality of amino acid positions among the naturally occurring hGH is described, it comprise strengthen the agonist activity, strengthen the proteolytic enzyme resistivity, make polypeptide be converted into the replacement of antagonist or the like and contained by term " hGH polypeptide ".
(for example, hGH) sequence comprises that (for example) comprises the naturally occurring hGH sequence of following modification: H18D, H21N, R167N, D171S, E174S, I179T to agonist GH.Referring to, for example, United States Patent (USP) the 5th, 849, No. 535, it is incorporated herein by reference.Other agonist hGH sequence comprises H18D, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; Or H18D, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A.Referring to, for example, United States Patent (USP) the 6th, 022, No. 711, it is incorporated herein by reference.The hGH polypeptide that comprises H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A replacement improves in the avidity of position I to the hGH acceptor.Referring to, for example, United States Patent (USP) the 5th, 854, No. 026, it is incorporated herein by reference.Protease resistant enhanced hGH sequence includes, but is not limited to comprise the hGH polypeptide of one or more aminoacid replacement in the C-D ring.In certain embodiments, replacement includes, but is not limited to R134D, T135P, K140A and its any combination.Referring to, for example, people such as Alam (1998) J.Biotechnol.65:183-190.
Human growth hormone's antagonist comprises that (for example) has replacement (for example, G120R, G120K, G120W, G120Y, G120F or G120E) and further comprise those human growth hormone's antagonists of following replacement: H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A sometimes at the G120 place.Referring to, for example, United States Patent (USP) the 6th, 004, No. 931, it is incorporated herein by reference.In certain embodiments, the hGH antagonist comprises at least one replacement in regional 106-108 or 127-129, and this makes GH can serve as antagonist.Referring to, for example, United States Patent (USP) the 6th, 608, No. 183, it is incorporated herein by reference.In certain embodiments, the hGH antagonist comprises the non-naturally encoded amino acid in the position II calmodulin binding domain CaM that is present in the hGH molecule that is connected with water-soluble polymers.In certain embodiments, the hGH polypeptide further comprises following replacement: H18D, H21N, R167N, K168A, D171S, K172R, E174S, I179T, and one be substituted in the G120 place (referring to, for example, United States Patent (USP) the 5th, 849, No. 535).
For naturally occurring human GH aminoacid sequence and the sophisticated naturally occurring GH aminoacid sequence and the naturally occurring mutant of complete total length, respectively referring to herein SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:3.In certain embodiments, any other sequence with SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or growth hormone polypeptides is identical substantially for GH polypeptide of the present invention (for example, hGH polypeptide).For instance, in certain embodiments, GH polypeptide of the present invention (for example, hGH polypeptide) has 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%, at least about 95% or identical with any other sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or growth hormone polypeptides at least about 99%.In certain embodiments, GH polypeptide of the present invention (for example, hGH polypeptide) has 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%, at least about 95% or identical with SEQ ID NO:2 at least about 99%.A large amount of naturally occurring hGH mutant are differentiated.These mutant comprise hGH-V (Seeburg, DNA 1:239 (1982); United States Patent (USP) the 4th, 446, No. 235, the 4th, 670, No. 393 and the 4th, 665, No. 180, it is incorporated herein by reference) and contain 20-kDa hGH (SEQ ID NO:3) (people such as Kostyo, the Biochem.Biophys.Acta 925:314 (1987) of disappearance of the residue 32-46 of hGH; Lewis, people such as U., J. Biol.Chem., 253:2679-2687 (1978)).At Igout, people such as A., NucleicAcids Res.17 (10): in 3998 (1989) placental growth hormone is described.In addition, reported by transcribing numerous hGH varients that back, translation back, secretion, metabolism processing and other physiological process produce, it comprises varient or 2 chain varients (Baumann, G., Endocrine Reviews 12:424 (1991)) that proteolysis splits into.At Lewis, people such as U.J., J. Biol.Chem.252:3697-3702 (1977), Brostedt, P. and Roos, P. has described among the Prep.Biochem.19:217-229 (1989) by the direct-connected hGH dimer of Cys-Cys disulfide linkage.The nucleic acid molecule of coding hGH mutant and sudden change hGH polypeptide is known by us and is included, but is not limited at United States Patent (USP) the 5th, 534 No. 617, the 5th, 580, No. 723, the 5th, 688, No. 666, the 5th, 750, No. 373, the 5th, 834, No. 250, the 5th, 834, No. 598, the 5th, 849, No. 535, the 5th, 854, No. 026, the 5th, 962, No. 411, the 5th, 955, No. 346, the 6th, 013, No. 478, the 6th, 022, No. 711, the 6th, 136, No. 563, the 6th, 143, No. 523, the 6th, 428, No. 954, the 6th, 451, No. 561, the 6th, those nucleic acid molecule that disclosed in 780, No. 613 and the U.S. Patent Application Publication case 2003/0153003 describedly are incorporated herein by reference with reference to case.
The commercial formulation of hGH is to sell with following title: Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono).
Term " hGH polypeptide " also comprises the pharmaceutically acceptable salt of the natural hGH of existence and prodrug, polymorphic form, hydrate, solvate, bioactive fragment, biological activity varient and the steric isomer of prodrug and salt, and the agonist of naturally occurring hGH, stand-in and antagonist varient and its polypeptide fusions.Comprise other amino acid whose fusions is all contained by term " hGH polypeptide " at aminoterminal, carboxyl terminal or two ends.The fusions that exemplary fusions includes, but is not limited to (for example) methionyl tethelin (wherein methionine(Met) is connected with N end by the hGH of the recombinant expressed generation of the mature form of the hGH that lacks secreting signal peptide or its part), is used for the fusions of purifying purpose (include, but is not limited to forms with polyhistidyl or affinity epitope fusions), form with the serum albumin binding peptide and with the fusions of serum protein (such as, serum albumin) formation.No. the 5th, 750,373, the United States Patent (USP) that is incorporated herein by reference is described a kind of the selection and is had the novel protein in conjunction with character (such as, tethelin) of change and the method for antibody fragment varient for its corresponding acceptor molecule.The carboxyl terminal structural domain that described method comprises the gene III coat protein that makes proteinic gene that coding pays close attention to and filobactivirus M13 merges.
Various reference disclose by polymkeric substance combination or glycosylation and come modified polypeptide.Term " hGH polypeptide " comprise with polymkeric substance (such as, PEG) in conjunction with and can comprise the polypeptide of one or more other derivative forms of halfcystine, Methionin or other residue.In addition, the hGH polypeptide can comprise connexon or polymkeric substance, wherein said connexon or polymkeric substance institute bonded amino acid can be according to alpha-non-natural amino acid of the present invention, maybe can utilize known technology in this technology (such as with Methionin or halfcystine coupling) combine with natural amino acids coding.
The polymkeric substance keying action of hGH polypeptide is reported.Referring to, for example, United States Patent (USP) the 5th, 849, No. 535, the 6th, 136, No. 563 and the 6th, 608, No. 183, it is incorporated herein by reference.United States Patent (USP) the 4th, 904, the polypeptide of No. 584 announcement PEGization Methionin disappearances, wherein at least one lysine residue has lacked or has replaced through any other amino-acid residue.WO 99/67291 discloses a kind of protein and PEG bonded method of making, and wherein at least one amino-acid residue on the protein lacks and under the condition that is enough to realize with protein bound protein contacted with PEG.WO99/03887 discloses the PEGization varient of the polypeptide that belongs to the tethelin superfamily, and wherein cysteine residues is replaced by the non-essential amino acid residue that is arranged in the polypeptide designation area.WO 00/26354 discloses a kind of method that produces the glycosylated polypeptides varient of allergenicity reduction, and described glycosylated polypeptides varient is when comparing with corresponding parent polypeptide, and it comprises at least one other glycosylation position.At least one other carbohydrate chain is introduced in the modification that the United States Patent (USP) that is incorporated herein by reference discloses granulocyte colony-stimulating factor (G-CSF) and other polypeptide for the 5th, 218, No. 092 when comparing with natural polypeptides with box lunch.
Term " hGH polypeptide " comprises that also glycosylated polypeptide takes place at any amino acid position place for glycosylation hGH and (but being not limited to), N-connects or the polypeptide of O-connection glycosylation form.The varient that contains the mononucleotide variation also is regarded as the biological activity varient of hGH polypeptide.In addition, also comprise splicing variants.Term " hGH polypeptide " also comprises by chemical mode and connects or (for example be expressed as any of fusion rotein or more than one GH, hGH) GH of other bioactive molecules of polypeptide or any other polypeptide, protein, carbohydrate, polymkeric substance, small molecules, connexon, ligand or any kind (for example, hGH) polypeptide heterodimer, homodimer, heteropolymer or homology polymer, and contain (for example) specificity disappearance or other modification but still keep bioactive polypeptide analog.
Except as otherwise noted (that is, and when explanation relatively be based on SEQ ID NO:1,3 or during other hGH sequence), otherwise (for example, all of the amino acid position in hGH) are mentioned the position that is based among the SEQ ID NO:2 to the GH that describes herein.Those skilled in the art will realize that, corresponding to SEQ ID NO:1,2,3 or any other GH sequence in the amino acid position of position can be in office what its GH is (for example, hGH) easily obtain identification in the molecule (such as, GH, or hGH fusions, varient, fragment etc.).For example, such as the sequence alignment program of BLAST can be used to compare and differentiate in protein corresponding to SEQ ID NO:1,2,3 or other GH sequence in the specific position of position.Herein with regard to SEQ ID NO:1,2,3 or other GH sequence with regard to the amino acid whose replacement, disappearance described add also be used to refer to describe herein or this technology in replacement, disappearance or interpolation on the corresponding position in known GH or hGH fusions, varient, the fragment etc., and obviously contained by the present invention.
Term " hGH polypeptide " or " hGH " are contained the hGH polypeptide that comprises one or more aminoacid replacement, interpolation or disappearance.HGH polypeptide of the present invention can comprise the modification carried out with one or more natural amino acids modification together with one or more alpha-non-natural amino acids.The exemplary replacement on a plurality of amino acid positions in the naturally occurring hGH polypeptide is described, described exemplary replacement include, but is not limited to the hGH polypeptide one or more biological activitys (strengthen the agonist activity such as (but being not limited to), increase polypeptide solvability, reduce proteolytic enzyme susceptibility, make polypeptide be converted into antagonist etc.) replacement, and described exemplary replacement contained in term " hGH polypeptide ".
Human GH antagonist includes, but is not limited in the position 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 and 127 places to have and replaces or 1 place has interpolation (promptly in the position, at N end) or its any combination (SEQ ID NO:2, SEQ ID NO:1,3 or any other GH sequence in corresponding amino acid) those antagonists.In certain embodiments, the hGH antagonist is in regional 1-5 (N end), 6-33 (spiral A), the 34-74 (zone between spiral A and the spiral B, the A-B ring), 75-96 (spiral B), the 97-105 (zone between spiral B and the spiral C, the B-C ring), 106-129 (spiral C), the 130-153 (zone between spiral C and the spiral D, C-D ring), comprise at least one replacement among 154-183 (spiral D), the 184-191 (C end), this makes GH can serve as antagonist.In other embodiments, non-naturally encoded amino acid whose exemplary aminoterminal zone that the position is included in spiral A and the residue within a part of spiral C incorporated into.In another embodiment, use such as non-naturally encoded aminoacid replacement G120 azido--L-phenylalanine or O-propargyl-L-tyrosine.In other embodiments, replacement of listing above and the other replacement that makes the hGH polypeptide become the hGH antagonist are made up.For example, the position in the position of being differentiated is with non-naturally encoded aminoacid replacement in this article, and introduces replacement (for example, G120R, G120K, G120W, G120Y, G120F or G120E) simultaneously at G120 place.In certain embodiments, the hGH antagonist comprises the non-naturally encoded amino acid in the hGH molecular receptor land of being present in that is connected with water-soluble polymers.
In certain embodiments, (for example, hGH) polypeptide further comprises bioactive interpolation, replacement or the disappearance of regulation and control GH or hGH polypeptide to GH.For example, add, replace or lack adjustable GH (for example, the character of hGH) one or more or activity.For example, add, replace or lack and to regulate and control (for example, the hGH) avidity of polypeptide receptor, regulation and control (including, but is not limited to strengthen or weaken) receptor dimerizationization GH, stablize receptor dimer, circulating half-life, the regulation and control treatment transformation period, regulation and control polypeptide stability, the cracking that regulation and control are undertaken by proteolytic enzyme, regulation and control dosage, regulation and control discharge or bioavailability, promote purifying, or improve or change specific dosing way.Similarly, (for example, hGH) polypeptide can comprise protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or polyhistidyl) or other sequence based on avidity (including, but is not limited to FLAG, polyhistidyl, GST etc.) or the link molecule (including, but is not limited to vitamin H) of detection (including, but is not limited to GFP), purifying or other characteristic of improving polypeptide to GH.
Homodimer, heterodimer, homology polymer and the heteropolymer that has connected also contained in term " hGH polypeptide ", and it includes, but is not limited to directly to be connected or to be connected or indirect those homodimers, heterodimer, homology polymer and the heteropolymer that is connected by connexon with natural amino acids coding side chain with identical or different non-naturally encoded amino acid side chain by non-naturally encoded amino acid side chain.Exemplary connexon include, but is not limited to the small molecules organic compound, have the water-soluble polymers of multiple length (such as, poly-(ethylene glycol) or poly-dextran) or have the polypeptide of all lengths.
" non-naturally encoded amino acid " refers to a kind of is not to be the amino acid of one of 20 kinds of common amino acids or pyrroles's Methionin or seleno-cysteine.Other term that can use with term " non-naturally encoded amino acid " synonym be " alpha-non-natural amino acid " and " amino acid of non-natural existence ".Term " non-naturally encoded amino acid " also include, but is not limited to by the modification (for example, posttranslational modification) of natural amino acids coding (including, but is not limited to 20 kinds of common amino acids or pyrroles's Methionin and seleno-cysteine) to occur but be not himself by the translation mixture the natural amino acid of incorporating in the growth polypeptide chain.The amino acid whose example that described non-natural exists includes, but is not limited to N-acetylglucosamine base-L-Serine, N-acetylglucosamine base-L-Threonine and O-phosphorylated tyrosine.
" aminoterminal modification group " refers to any molecule that can be connected with the aminoterminal of polypeptide.Similarly, " carboxyl terminal modification group " refers to any molecule that can be connected with the carboxyl terminal of polypeptide.End modified group includes, but is not limited to various water-soluble polymerss, peptide or such as sero-abluminous protein or other parts that the serum half-life of peptide is prolonged.
Term " functional group ", " active part ", " activating group ", " leavings group ", " reactive moieties ", " chemically reactive group " and " chemical reactivity part " are the uniqueness that is used to refer to molecule, confirmable part or unit in this technology neutralization in this article.Identical a bit and be used for representing to carry out some function in this article or have the part of some molecules active and that easily react with other molecule in the meaning of term described in the chemical technology.
Term " key " or " connexon " are to be used to refer to group or the key that forms and be generally covalent linkage usually owing to chemical reaction in this article.The key of hydrolysis-stable means and is substantially stable in water and (includes, but is not limited under physiological condition) in one long-term period the key that (perhaps even indefinitely) do not react with water under effective pH value.Hydrolytically unstable or degradable key mean in water or degradable key in the aqueous solution (comprising for example blood).Enzymatic instability or degradable key mean can be by the key of one or more enzyme liberating.Such as in this technology understanding, PEG and related polymer can comprise degradable key in main polymer chain or in the connexon group between the one or more functional end-group of main polymer chain and polymer molecule.For example, the ester bond that forms by the pure radical reaction on PEG carboxylic acid or activated PEG carboxylic acid and the biologically active agent generally under physiological condition hydrolysis discharge reagent.The degradable key of other hydrolysis includes, but is not limited to carbonic acid ester bond; Imine linkage by amine and aldehyde reaction generation; React the phosphoric acid ester bond that forms by alcohol and bound phosphate groups; Hydrazone key for the reaction product of hydrazides and aldehyde; Be the acetal bonds of aldehyde with the reaction product of alcohol; Be the original acid ester key of manthanoate with the reaction product of alcohol; By including, but is not limited at polymkeric substance (such as, the PEG) peptide bond that forms of the carboxyl of Mo Duan amido and peptide; With the oligonucleotide key that forms by the 5 ' hydroxyl that includes, but is not limited at the phosphoramidite group of polymer ends and oligonucleotide.
Term " bioactive molecules ", " biologically-active moiety " or " biologically active agent " when using in this article, mean any physical properties that can influence biosystem, path, molecule or biochemical property or with the relevant interactional any material of organism (including, but is not limited to virus, bacterium, phage, transposon, Protein virus, insect, fungi, plant, animal and human's class)." bioactive molecules " includes, but is not limited to be intended to be used to any material of the good order and condition of the health diagnosing, cure, alleviate, treat or prevent the disease of human or other animal or can strengthen the mankind or animal in addition or spirit specifically, as used herein.The example of bioactive molecules includes, but is not limited to peptide, protein, enzyme, small-molecule drug, hard medicine, soft medicine, carbohydrate, inorganic atoms or molecule, dyestuff, lipid, nucleosides, radionuclide, oligonucleotide, toxin, cell, virus, liposome, particulate and micelle.The classification that is suitable for the biologically active agent that uses for the present invention includes, but is not limited to medicine, prodrug, radionuclide, photographic developer, polymkeric substance, microbiotic, mycocide, antiviral agent, anti-inflammatory agents, antineoplastic agent, cardiovascular agents, antianxiety agent, hormone, somatomedin, steroid medicament, is derived from toxin of microorganism or the like.
" double functional copolymer " refers to the polymkeric substance that comprises two discrete functional groups, and described functional group can specifically react to form covalent linkage or non covalent bond with other parts (including, but is not limited to amino acid side group).Have one and can can be used to form the binding substances that comprises first biologically active components, difunctionality connexon and second biologically active components with the difunctionality connexon of the group of radical reaction on second biological components with the functional group of radical reaction on the particular organisms active ingredient and another.The many programs and the connexon molecule that are used to all cpds is connected with peptide are known.Referring to, for example, No. the 4th, 671,958, No. the 188th, 256, European patent application, United States Patent (USP), the 4th, 659, No. 839, the 4th, 414, No. 148, the 4th, 699, No. 784, the 4th, 680, No. 338 and the 4th, 569, No. 789, it is incorporated herein by reference." polyfunctional poly compound " refers to the polymkeric substance that comprises two or more discrete functional groups, and described functional group can specifically react to form covalent linkage or non covalent bond with other parts (including, but is not limited to amino acid side group).Double functional copolymer or polyfunctional poly compound can be any length or molecular weight wanted, and can be selected so that (for example, hGH) specific desired interval or the conformation between the one or more molecule that is connected of molecule with GH to be provided.
At substituting group is that it contains the chemically identical substituting group that obtains by writing structure from right to left equally under the conventional chemical formula of from left to right writing by it situation about illustrating, for example, and structure-CH 2O-is equal to structure-OCH 2-.
Term " substituting group " includes, but is not limited to " non-interfering substituting group "." non-interfering substituting group " is for producing those groups of stable compound.Suitable non-interfering substituting group or group include, but is not limited to halogen, C 1-C 10Alkyl, C 2-C 10Thiazolinyl, C 2-C 10Alkynyl, C 1-C 10Alkoxyl group, C 1-C 12Aralkyl, C 1-C 12Alkaryl, C 3-C 12Cycloalkyl, C 3-C 12Cycloalkenyl group, phenyl, the phenyl that is substituted, toluyl, xylyl, xenyl, C 2-C 12Alkoxyalkyl, C 2-C 12Alkoxy aryl, C 7-C 12Aryloxy alkyl, C 7-C 12Oxygen Ji Fangji, C 1-C 6Alkyl sulphinyl, C 1-C 10Alkyl sulphonyl ,-(CH 2) m-O-(C 1-C 10Alkyl) (wherein m is 1 to 8), aryl, the aryl that is substituted, the alkoxyl group that is substituted, fluoroalkyl, heterocyclic radical, the heterocyclic radical that is substituted, 4-nitro alkyl ,-NO 2,-CN ,-NRC (O)-(C 1-C 10Alkyl) ,-C (O)-(C 1-C 10Alkyl), C 2-C 10Alkyl alkylthio base ,-C (O) O-(C 1-C 10Alkyl) ,-OH ,-SO 2,=S ,-COOH ,-NR 2, carbonyl ,-C (O)-(C 1-C 10Alkyl)-CF 3,-C (O)-CF 3,-C (O) NR 2,-(C 1-C 10Aryl)-S-(C 6-C 10Aryl) ,-C (O)-(C 1-C 10Aryl) ,-(CH 2) m-O-(CH 2) m-O-(C 1-C 10Alkyl) (wherein each m is 1 to 8) ,-C (O) NR 2,-C (S) NR 2,-SO 2NR 2,-NRC (O) NR 2,-NRC (S) NR 2, its salt group or the like.Each R is H, alkyl or the alkyl that is substituted, aryl or the aryl that is substituted, aralkyl or alkaryl as used herein.
Term " halogen " comprises fluorine, chlorine, iodine and bromine.
Term " alkyl ", except as otherwise noted, otherwise it is to mean to can be fully saturated, monounsaturated or polyunsaturated and can comprise having appointment carbonatoms (that is C, individually or as another substituent part 1-C 10Mean 1 to 10 carbon atom) divalence and straight or branched or cyclic hydrocarbon group or its combination of multivalence group.The example of saturated hydrocarbyl includes, but is not limited to following group: the homologue of methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, the tertiary butyl, isobutyl-, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, (for example) n-pentyl, n-hexyl, n-heptyl, n-octyl and isomer or the like.Unsaturated alkyl is for having one or more pair of key or triple-linked group.The example of unsaturated alkyl includes, but is not limited to vinyl, 2-propenyl, butenyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), ethynyl, 1-proyl and 3-proyl, 3-butynyl and high-carbon homologue and isomer.Except as otherwise noted, otherwise term " alkyl " is also intended to comprise following those alkyl derivatives than specific definition, such as " assorted alkyl ".The alkyl that will be limited to alkyl is called " pure alkyl ".
Term " alkylene ", its individually or as another substituent part be mean be derived from alkane following (but being not limited to) by structure-CH 2CH 2-with-CH 2CH 2CH 2CH 2-illustrated divalent group and further comprising describes below those groups into " assorted alkylene ".Usually, alkyl (or alkylene) will have 1 to 24 carbon atom, those have 10 carbon atoms or still less the group of carbon atom be the specific embodiments of method and composition as herein described." low-carbon alkyl " or " lower alkylene " is for generally having 8 or the short alkyl or the alkylene of chain of carbon atom still less.
Term " alkoxyl group ", " alkylamino " and " alkyl sulfenyl " (or sulfenyl alkoxyl group) are to use with its conventional meaning, and refer to those alkyl that are connected with the rest part of molecule by Sauerstoffatom, amino or sulphur atom respectively.
Term " assorted alkyl ", except as otherwise noted, otherwise it is to mean to comprise definite carbonatoms and at least one is selected from heteroatomic stable straight or branched or cyclic hydrocarbon group or its combination of the group that is made up of O, N, Si and S individually or with the combination of another term, wherein nitrogen-atoms and sulphur atom optionally can be oxidized and nitrogen heteroatom optionally can be by quaternized.Heteroatoms O, N can place any interior location place of assorted alkyl or the position that places alkyl to be connected with the rest part of molecule with S and Si.Example includes, but is not limited to-CH 2-CH 2-O-CH 3,-CH 2-CH 2-NH-CH 3,-CH 2-CH 2-N (CH 3)-CH 3,-CH 2-S-CH 2-CH 3,-CH 2-CH 2,-S (O)-CH 3,-CH 2-CH 2-S (O) 2-CH 3,-CH=CH-O-CH 3,-Si (CH 3) 3,-CH 2-CH=N-OCH 3With-CH=CH-N (CH 3)-CH 3Two heteroatomss can be successive at the most, such as ,-CH 2-NH-OCH 3With-CH 2-O-Si (CH 3) 3Similarly, term " assorted alkylene ", it is to mean following (but being not limited to) of being derived from assorted alkyl by-CH individually or as another substituent part 2-CH 2-S-CH 2-CH 2-with-CH 2-S-CH 2-CH 2-NH-CH 2-illustrated divalent group.For assorted alkylene, identical or different heteroatoms also can be positioned at an one or both ends (including, but is not limited to alkylene oxygen base, alkylenedioxy, alkylene amino, alkylene diamino, amino oxygen base alkylene or the like) of chain.In addition, for alkylene and assorted alkylene linking group, the presentation direction of the formula of linking group does not also mean that the orientation of linking group.For example, formula-C (O) 2R '-expression-C (O) 2R '-with-R ' C (O) 2-.
Term " cycloalkyl " and " Heterocyclylalkyl ", except as otherwise noted, otherwise its individually or with the combination of other term be the annular form of representing " alkyl " and " assorted alkyl " respectively.Thereby that cycloalkyl or Heterocyclylalkyl can comprise is saturated, part is unsaturated and complete undersaturated ring key.In addition, for Heterocyclylalkyl, heteroatoms can be positioned at the position that heterocycle is connected with the rest part of molecule.The example of cycloalkyl includes, but is not limited to cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, suberyl or the like.The example of Heterocyclylalkyl includes, but is not limited to 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl or the like.In addition, dicyclo and tricyclic structure contained in described term.Similarly, term " heterocycle alkylene ", it is to mean the divalent group that is derived from Heterocyclylalkyl individually or as another substituent part, and term " cyclic alkylene ", and it is to mean the divalent group that is derived from cycloalkyl individually or as another substituent part.
Term " water-soluble polymers " refers to any polymkeric substance that dissolves in the water-based solvent as used herein.Water-soluble polymers and GH are (for example, hGH) connection of polypeptide can cause following change: include, but is not limited to respect to the unmodified form, serum half-life obtains prolonging or regulation and control, or the treatment transformation period obtains prolonging or regulation and control, immunogenicity obtains regulation and control, physics association characteristic such as aggregate and polymer formation obtains regulation and control, changes receptors bind and changes receptor dimerizationization or multimerization.Water-soluble polymers can have the biological activity of himself or can not have the biological activity of himself, and can be with GH (for example as connexon, hGH) (GH (for example, hGH) polypeptide or one or more bioactive molecules) that includes, but is not limited to one or more connects with other material.Suitable polymers includes, but is not limited to polyoxyethylene glycol, polyoxyethylene glycol propionic aldehyde, its single C 1-C 10Alkoxyl group or aryloxy derivative (are described in United States Patent (USP) the 5th, 252, in No. 714, described patent is incorporated herein by reference), mono methoxy polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, the divinyl ether MALEIC ANHYDRIDE, N-(2-hydroxypropyl)-Methacrylamide, dextran, the glucan derivative that comprises T 500, polypropylene glycol, polyoxytrimethylene/ethylene oxide copolymer, the polyoxyethylene polyvalent alcohol, heparin, heparin fragment, polysaccharide, oligosaccharides, glycan, Mierocrystalline cellulose and derivatived cellulose (including, but is not limited to methylcellulose gum and carboxymethyl cellulose), starch and starch derivative, polypeptide, polyalkylene glycol and its derivative, the multipolymer of polyalkylene glycol and its derivative, polyvinyl ethyl ether and alpha-beta-poly-[(2-hydroxyethyl)-DL-l-asparagine or the like or its mixture.The example of described water-soluble polymers includes, but is not limited to polyoxyethylene glycol and serum albumin.
Term " polyalkylene glycol " or " poly-(alkene glycol) " refer to polyoxyethylene glycol (poly-(ethylene glycol)), polypropylene glycol, polytetramethylene glycol and its derivative as used herein.Term " polyalkylene glycol " and/or " polyoxyethylene glycol " are contained linearity and branched polymers and molecular-weight average between 0.1kDa and 100kDa.For example, in goods catalogue, enumerated other one exemplary embodiment such as the commercial supplier of the catalogue " Polyethylene Glycol and Derivatives for BiomedicalApplications " (2001) of Shearwater Corporation.
Except as otherwise noted, otherwise term " aryl " means how unsaturated aromatic hydrocarbon substituting group, and it can be monocycle or condenses together or covalently bound many rings (including, but is not limited to 1 to 3 ring).Term " heteroaryl " refers to and contains 1 to 4 heteroatomic aryl that is selected from N, O and S (or ring), wherein nitrogen-atoms and sulphur atom optionally can be oxidized and nitrogen-atoms optionally can be by quaternized.Heteroaryl can be connected with the rest part of molecule by heteroatoms.The limiting examples of aryl and heteroaryl comprises phenyl, the 1-naphthyl, the 2-naphthyl, the 4-xenyl, the 1-pyrryl, the 2-pyrryl, the 3-pyrryl, the 3-pyrazolyl, the 2-imidazolyl, the 4-imidazolyl, pyrazinyl, the 2-oxazolyl, the 4-oxazolyl, 2-phenyl-4-oxazolyl, the 5-oxazolyl, the 3-isoxazolyl, the 4-isoxazolyl, the 5-isoxazolyl, the 2-thiazolyl, the 4-thiazolyl, the 5-thiazolyl, the 2-furyl, the 3-furyl, the 2-thienyl, the 3-thienyl, the 2-pyridyl, the 3-pyridyl, the 4-pyridyl, the 2-pyrimidyl, the 4-pyrimidyl, the 5-benzothiazolyl, purine radicals, the 2-benzimidazolyl-, the 5-indyl, the 1-isoquinolyl, the 5-isoquinolyl, the 2-quinoxalinyl, the 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.Each person's substituting group is to be selected from the substituent group that accepts described below in above-mentioned aryl and the heteroaryl ring system.
For for simplicity, term " aryl " is when comprising aryl and heteroaryl as defined above when being used in combination with other term (including, but is not limited to aryloxy, aryl sulphur oxygen base, arylalkyl).Thereby, term " arylalkyl " is intended to those groups (including, but is not limited to phenmethyl, styroyl, pyridylmethyl or the like) of comprising that aryl is connected with alkyl, and it comprises that carbon atom (including, but is not limited to methylene radical) has been (for example) Sauerstoffatom (including, but is not limited to phenoxymethyl, 2-pyridyl oxygen ylmethyl, 3-(1-naphthyl oxygen base) propyl group or the like) institute's those alkyl of metathetical.
Above each term (including, but is not limited to " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ") be intended to comprise shown in the form that is substituted of group and the form that is unsubstituted.The exemplary substituting group of all kinds of groups is provided below.
The substituting group of alkyl and assorted alkyl (comprising those groups that often are called alkylene, thiazolinyl, assorted alkylene, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) can be the one or more group in the group of the following group of multiple being selected from (but being not limited to) :-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R  ,-OC (O) R ' ,-C (O) R ' ,-CO 2R ' ,-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 " ,-NRSO 2R ' ,-CN and-NO 2, quantity changes to (2m '+1) scope 0, and wherein m ' is the total number of carbon atoms in the described group.R ', R ", R  and R " " aryl that independently refer to hydrogen, the assorted alkyl that is substituted or is unsubstituted separately, is substituted or is unsubstituted (include, but is not limited to replace aryl), alkyl, alkoxyl group or the thioalkoxy group or the arylalkyl that are substituted or are unsubstituted through 1-3 halogen atom.For example, when compound of the present invention comprises a plurality of R group, as R ', R ", R  and R " " when a plurality of groups in the group exist its each selected independently naturally, each R group is selected independently." when being connected with same nitrogen-atoms, they can be combined to form 5 yuan of rings, 6 yuan of rings or 7 yuan of rings with described nitrogen-atoms as R ' and R.For example ,-NR ' R " is intended to include, but is not limited to 1-pyrrolidyl and 4-morpholinyl.According to discussing above substituent, be understood by those skilled in the art that term " alkyl " is intended to comprise and comprises and the non-hydrogen group group of the carbon atom of bond mutually, (includes, but is not limited to-CF such as alkylhalide group 3With-CH 2CF 3) and acyl group (include, but is not limited to-C (O) CH 3,-C (O) CF 3,-C (O) CH 2OCH 3Or the like).
Be similar to about the described substituting group of alkyl, the substituting group of aryl and heteroaryl can change and be to be selected from (but being not limited to) following group: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R  ,-OC (O) R ' ,-C (O) R ' ,-CO 2R ' ,-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 " ,-NRSO 2R ' ,-CN and-NO 2,-R ' ,-N 3,-CH (Ph) 2, fluorine-based (C 1-C 4) alkoxyl group and fluorine-based (C 1-C 4) alkyl, quantity changes in 0 available valency sum scope to the aromatic ring system; And wherein R ', R ", R  and R " " are independently to be selected from hydrogen, alkyl, assorted alkyl, aryl and heteroaryl.For example, when compound of the present invention comprises a plurality of R group, as R ', R ", R  and R " " when a plurality of groups in the group exist its each selected independently naturally, each R group is selected independently.
Term " through the serum half-life of regulation and control " means positivity or the negativity variation of the circulating half-life of modified hGH with respect to its unmodified form as used herein.Serum half-life is to measure by each time point sample of blood, drawn after hGH is given in throwing and the molecular conecentration of measuring in each sample.The mutual relationship of serum-concentration and time makes serum half-life to calculate.The serum half-life that wish to prolong has at least about twice, still (for example) gratifying dosage regimen to be provided or to avoid prolongation less under the situation of toxic effect at it may be useful.In certain embodiments, prolongation amount is at least about three times, at least about five times or at least about ten times.
Term " through the treatment transformation period of regulation and control " means positivity or the negativity variation of the transformation period of the modified hGH that treats significant quantity with respect to its unmodified form as used herein.The treatment transformation period is to measure by the pharmacokinetics and/or the drug effect character of each point in time measurement molecule after dispensing.Wish that the treatment transformation period that prolongs can provide specific useful dosage regimen, specific useful total dose or avoid ill effect.In certain embodiments, the treatment transformation period that prolongs by the enhancing of enhanced effectiveness, decorating molecule and its target spot or weaken combine, pass through enzyme (such as, proteolytic enzyme) enhancing of the molecular breakdown of carrying out or weaken, or the enhancing (adding) of another parameter of unmodified molecule or the mechanism of action or weaken (little) and produce.
Term " separation " is when being applied to nucleic acid or protein, it represents that described nucleic acid or protein do not contain at least some cellular components associating with it under native state, or represents that described nucleic acid or protein have been concentrated to the level greater than its concentration that or in vitro produces in vivo.It can be homogeneous state.Separated material can be drying regime or leather hard, perhaps is in solution (including, but is not limited to the aqueous solution) state.It can be the component of the medical composition that comprises other pharmaceutically acceptable supporting agent and/or vehicle.Purity and uniformity typically use such as the technique of analytical chemistry of polyacrylamide gel electrophoresis or high performance liquid chromatography and measure.For the protein that is present in the main material in the preparation is purifying substantially.Specifically, separated gene is also to encode from the side joint gene to be different from the proteinic open reading frame separation of the gene of being paid close attention to.Term " purifying " expression nucleic acid or protein produce in running gel and are essentially one band.Exactly, it can mean nucleic acid or proteinic purity and is at least 85%, at least 90%, at least 95%, at least 99% or bigger purity.
Term " nucleic acid " refers to deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and its polymkeric substance that is sub-thread chain form or double-stranded chain form.Unless otherwise specifically limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in term, its have with reference nucleic acid similar combine character and to be similar to the mode metabolism of naturally occurring Nucleotide.Unless special restriction is arranged in addition, otherwise described term also refers to the oligonucleotide analogs of the analogue (thiophosphatephosphorothioate, phosphoramidate or the like) that comprises PNA (peptide nucleic acid(PNA)), is used for the DNA of antisense technology.Unless otherwise noted, otherwise the sequence that specific nucleotide sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and clearly indicated.Especially, degenerate codon replaces and can reach by producing following sequence, one of them or more than one the 3rd position of selected (or all) codons replace (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by mixing base and/or Hypoxanthine deoxyriboside residue; People such as Ohtsuka, J. Biol.Chem.260:2605-2608 (1985); People such as Rossolini, Mol.Cell.Probes 8:91-98 (1994)).
Term " polypeptide ", " peptide " and " protein " commutative in this article use are to be used to refer to the polymkeric substance of amino-acid residue.That is, be equally applicable to the description and the proteinic description of peptide at the description of polypeptide, and vice versa.Described term is applicable to that naturally occurring aminoacid polymers and one of them or more than one amino-acid residue are non-naturally encoded amino acid whose aminoacid polymers.The amino acid chain with any length contained in term as used herein, and it comprises full length protein, and wherein amino-acid residue is to connect by the covalency peptide bond.
Term " amino acid " refers to amino acid naturally occurring and that non-natural exists, and to be similar to amino acid analogue and the amino acid analog thing that naturally occurring amino acid whose mode works.Natural amino acids coding is 20 kinds of common amino acid (L-Ala, arginine, asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan) and pyrroles's Methionin and seleno-cysteine.Amino acid analogue refers to the compound with basic chemical structure identical with naturally occurring amino acid, promptly, have the α carbon compound with hydrogen, carboxyl, amino and R group bond, such as, homoserine, nor-leucine, methionine sulfoxide, methionine(Met) methyl sulfonium.Described analogue has modified R group (such as, nor-leucine) or modified peptide main chain, but keeps the basic chemical structure identical with naturally occurring amino acid.
Amino acid can be mentioned by its common known 3 letter characters or by 1 letter character of being recommended by IUPAC-IUBBiochemical Nomenclature Commission in this article.Similarly, Nucleotide can be mentioned by its common received single-letter code.
" through the conservative varient of modifying " is applicable to amino acid and nucleotide sequence.For specific nucleotide sequence, " through the conservative varient of modifying " refers to those nucleic acid of the identical or substantially the same aminoacid sequence of coding, or not during encoding amino acid sequence, refers to substantially the same sequence at nucleic acid.Since the degeneracy of genetic code, any appointment protein of the nucleic acid encoding that many functions are identical.For example, codon GCA, GCC, GCG and GCU coded amino acid L-Ala all.Thereby, be each position of determining by codon at L-Ala, described codon can be changed into described any corresponding codon and not change encoded polypeptide.It is " static variation " that described nucleic acid changes, and it is a kind of variation of conservative modification.Each nucleotide sequence of coded polypeptide is also described each possible static variation of nucleic acid herein.Those skilled in the art will realize that each codon in the nucleic acid (except that only being the AUG of the codon of methionine(Met) and only for the TGG of the codon of tryptophane usually usually) can be by modification with the identical molecule of generation function.Therefore, the various static variation of nucleic acid encoding lies in described each sequence.
About aminoacid sequence, those skilled in the art will realize that, change, add or remove single amino acids in the coded sequence or amino acid whose indivedual replacements, disappearance or the interpolation to nucleic acid, peptide, polypeptide or protein sequence of little per-cent is a kind of " conservative variation of modifying ", wherein said change causes that the similar amino acid of amino acid whose disappearance, amino acid whose interpolation or chemical property is to amino acid whose replacement.It is known to those of ordinary skill in the art that intimate amino acid whose conservative replacement table is provided.So through the conservative varient of modifying be remove polymorphic variant of the present invention, plant between homologue and the allelotrope varient and do not get rid of homologue and allelotrope between polymorphic variant of the present invention, kind.
It is known to those of ordinary skill in the art that intimate amino acid whose conservative replacement table is provided.Below eight groups of amino acid that respectively contain promising conservative replacement each other:
1) L-Ala (A), glycine (G);
2) aspartic acid (D), L-glutamic acid (E);
3) asparagine (N), glutamine (Q);
4) arginine (R), Methionin (K);
5) Isoleucine (I), leucine (L), methionine(Met) (M), Xie Ansuan (V);
6) phenylalanine (F), tyrosine (Y), tryptophane (W);
7) Serine (S), Threonine (T); With
8) halfcystine (C), methionine(Met) (M)
(referring to, for example, Creighton, Proteins:Structures and Molecular Properties, W H Freeman﹠amp; Co.; Second edition (in December, 1993))
Under the situation of two or more nucleic acid or peptide sequence, term " identical " or per-cent " identity " refer to two or more and are identical sequence or subsequence.When comparing on comparison window or on the designated area and comparing maximum correspondence, as use a kind of algorithm in the following sequence comparison algorithm (or available other algorithm of those skilled in the art) or record by manual comparison and visual inspection, as infructescence have for the per-cent of identical amino-acid residue or Nucleotide (promptly, identity at least about 60% is arranged on the regulation zone, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identity), so described sequence is exactly " identical substantially ".This definition also refers to the complementary sequence of cycle tests.Identity can be present on the zone that length is at least about 50 amino acid or Nucleotide, or is present on the zone that length is 75-100 amino acid or Nucleotide, or when not stipulating, crosses over the whole sequence of polynucleotide or polypeptide.
For sequence relatively, a common sequence is served as the reference sequences of comparing with cycle tests.When using sequence comparison algorithm, in cycle tests and reference sequences input computer, (in case of necessity) specifies subsequence coordinate and specified sequence algorithm routine parameter.The program parameter of acquiescence can be used, maybe substituting parameter can be specified.Sequence comparison algorithm calculates the sequence identity per-cent of cycle tests with respect to reference sequences based on program parameter subsequently.
As used herein " comparison window " comprise relate to have be selected from by 20 to 600, be generally about 50 to about 200, be more typically any one in about 100 to about 150 groups that form and adjoin the section of figure place, wherein two sequences by after the best comparison, can with a sequence with have the identical reference sequences that adjoins figure place and compare.The sequence alignment method that is used for comparison is known to those of ordinary skill in the art.Be used for the optimal sequence comparison of comparison, can include, but is not limited to local homology's algorithm by Smith and Waterman (1970) Adv.Appl.Math.2:482c, homology alignment algorithm by Needleman and Wunsch (1970) J. Mol.Biol.48:443, searching similarity method by Pearson and Lipman (1988) Proc.Nat ' I.Acad.Sci.USA 85:2444, (Wisconsin Genetics Software Package is implemented in computerize by these algorithms, Genetics Computer Group, 575 ScienceDr., Madison, GAP among the WI, BESTFIT, FASTA and TFASTA), or by manual comparison and visual inspection (referring to, for example, people such as Ausubel, Current Protocols in Molecular Biology (1995 supplementary issue)) carry out.
An example that is suitable for measuring the algorithm of sequence identity and sequence similarity per-cent is BLAST and BLAST 2.0 algorithms, and it is described in respectively among people (1990) J.Mol.Biol.215:403-410 such as people such as Altschul (1997) Nuc.Acids Res.25:3389-3402 and Altschul.Being used to carry out software that BLAST analyzes can be by can be at www. Ncbi.nlm.nih.govThe National Center for Biotechnology Information that network address obtains and open acquisition.The sensitivity and the speed of BLAST algorithm parameter W, T and X decision comparison.BLASTN program (being used for nucleotide sequence) uses that word length (W) is 11, expected value (E) is 10, the comparison of M=5, N=-4 and two strands of chains is as default value.With regard to aminoacid sequence, the BLASTP program use word length be 3 and expected value (E) be 10 as default value, and BLOSUM62 keeps the score that matrix (referring to Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.USA 89:10915) uses that comparison value (B) is 50, expected value (E) is 10, the comparison of M=5, N=-4 and two strands of chains is as default value.The BLAST algorithm is normally carried out when wave filter cuts out at " low complex degree ".
The BLAST algorithm is also carried out the statistical study (referring to, for example, Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5787) of two similaritys between the sequence.A kind of the measuring of the similarity that is provided by the BLAST algorithm is minimum and probability (P (N)), and it represents that coupling between two Nucleotide or the aminoacid sequence is with occurrent probability.For example, if minimum in the comparison of test nucleic acid and reference nucleic acid and probability less than about 0.2, or less than about 0.01, or less than about 0.001, it is similar to reference sequences to look described nucleic acid so.
Phrase " optionally (or specifically) and ... hybridization " refers to when a specific nucleotide sequence and is present in complex mixture (including, but is not limited to total cell or library DNA or RNA) when middle, under stringent hybridization condition, molecule only combines with described nucleotide sequence, compound or hybridization.
Phrase " stringent hybridization condition " refers under low ionic strength and hot conditions as known in the art, and the sequence of DNA, RNA, PNA or other nucleic acid mimics or its combination is hybridized.Usually, under stringent condition, probe will with its target sequence hybridization in the complex mixture (including, but is not limited to total cell or library DNA or RNA) of nucleic acid, but not with described complex mixture in other sequence hybridization.Stringent condition be sequence dependent and will be under different situations for different.Long sequence is hybridized under higher temperature especially.The extensive guide of nucleic acid hybridization is found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology--Hybridization with Nucleic Probes is in " Overview of principles of hybridization andthe strategy of nucleic acid assays " (1993).Usually, stringent condition is chosen to be than at the ionic strength of determining, the thermal melting point (T of particular sequence under the pH value m) low about 5-10 ℃.T mBe under equilibrium state 50% with the hybridization of target complementary probe and target sequence (because of at T mThe excessive existence of following target sequence is so take 50% probe under equilibrium state) time temperature (under ionic strength, pH value and the nucleic acid concentration of determining).Stringent condition can be under 7.0 to 8.3 pH value, salt concn is less than about 1.0M sodium ion, be generally about 0.01 to 1.0M Na ion concentration (or other salt) and for short probe (including, but is not limited to 10 to 50 Nucleotide), temperature is at least about 30 ℃ and for long probe (including, but is not limited to greater than 50 Nucleotide), temperature is at least about those stringent conditions of 60 ℃.Stringent condition also can be realized such as the destabilizing agent of methane amide by adding.For selective cross or specific hybrid, positive signal can be at least 2 times of background hybridizations, optionally is 10 times of background hybridizations.Exemplary stringent hybridization condition can be as follows: 50% methane amide, 5X SSC and 1%SDS, cultivate down at 42 ℃, or 5X SSC, 1%SDS, cultivate down, and under 65 ℃, in 0.2X SSC and 0.1%SDS, wash at 65 ℃.Described washing can be carried out more than 5,15,30,60,120 or 120 minutes.
Term " eukaryote " refers to and belongs to the organism that territory (phylogeneticdomain Eucarya) takes place in eukaryote kind system as used herein, such as, animal (including, but is not limited to Mammals, insect, Reptilia, bird etc.), ciliate, plant (including, but is not limited to monocotyledons, dicotyledons, algae etc.), fungi, yeast, flagellate, microparticle insect order, protobiont etc.
Term " non-eukaryote " refers to non-eucaryon organism as used herein.For example, non-eucaryon organism can belong to eubacterium and (include, but is not limited to bacillus coli (Escherichia coli), thermophilic bacterium (Thermus thermophilics), bacstearothermophilus (Bacillus stearothermophilus), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida) etc.) plant system the territory takes place, or ancient bacterium (includes, but is not limited to Methanococcus jannaschii (Methanococcus jannaschii), horizontal river virus (Methanobacterium thermoautotrophicum), the Halobacterium (Halobacterium) of having a liking for the richly endowed bacterium of salt (Haloferax volcanii) and having a liking for salt bacillus specie (Halobacterium species) NRC-1 such as Wo Shi, the ancient green-ball bacterium (Archaeoglobus fulgidus) of glimmering, fierce fireball bacterium (Pyrococcus furiosus), hole Yue Shi fireball bacterium (Pyrococcus horikoshii), the quick hot bacterium of gas of thermophile bacteria (Aeuropyrum pernix) etc.) plant system the territory takes place.
Term " person under inspection " refers to animal as used herein, is Mammals in certain embodiments, and is human in other embodiments, and it is the object of treatment, observation or experiment.
Term " significant quantity " refers to the amount of the modified non-natural amino acid polypeptides that gives of throwing as used herein, and it will alleviate one or more symptom of disease, illness or the deficiency disorder of just treating to a certain extent.Can throw and give the composition that contains modified non-natural amino acid polypeptides as herein described and be used for preventative, enhancing property and/or curative treatment.
Term " enhancing " means effectiveness or the time length that increases or prolong the effect of wanting.Therefore, just strengthen the effect of therapeutical agent, term " enhancing " refers to aspect effectiveness or time length, increases or prolong the ability of other therapeutical agent to the effect that system rose." enhancing significant quantity " refers to the amount that is enough to strengthen the effect of another therapeutical agent in the system of wanting as used herein.When being used for the patient, the amount that is effective to this purposes will be decided on the severity of disease, deficiency disorder or illness and the course of disease, previous treatment, patient's healthy state with to the reaction of medicine and treatment doctor's judgement.
Term " modified " refers to any change to specifying polypeptide to do as used herein, such as, change length, amino acid sequence of polypeptide, chemical structure, the translation modification or the posttranslational modification altogether of polypeptide.Term " (modified) " form means the polypeptide of being discussed and is optionally modified, that is, the polypeptide of being discussed can be modified or do not modified.
Term " through posttranslational modification " refers to any modification natural or that alpha-non-natural amino acid has been done described amino acid after it has incorporated in the polypeptide chain.Described term contain (only for instance) altogether in vitro modification of in vivo the modifying of translation, translation altogether (such as, in cell free translation system), the in vitro modification after in vivo the modifying and translating after the translation.
In prophylactic application, the composition that will contain modified non-natural amino acid polypeptides is thrown to give and is easy to suffer from specified disease, deficiency disorder or illness or not so emits the patient that the danger of suffering from specified disease, deficiency disorder or illness is arranged.Described amount is defined as " prevention significant quantity ".In this kind purposes, accurate amount is also decided on patient's state of health, body weight or the like factor.The those skilled in the art is to determining that by normal experiment (for example, dosage escalation clinical trial) described prevention significant quantity has abundant understanding.
Term " through what protect " refers to " protecting group " or the existence of part that prevents that chemical reactivity functional group from reacting under some reaction conditions.Protecting group will be looked the type of protected chemically reactive group and be changed.For example, if chemically reactive group is amine or hydrazides, protecting group can be selected from the group of tertbutyloxycarbonyl (t-Boc) and 9-fluorenyl methoxy carbonyl (Fmoc) so.If chemically reactive group is a mercaptan, protecting group can be adjacent pyridyl disulfide so.If chemically reactive group is the group such as the carboxylic acid of butyric acid or propionic acid, or hydroxyl, protecting group can be phenmethyl or such as the alkyl of methyl, ethyl or the tertiary butyl so.Known other protecting group in this technology comprises the group to photo-labile such as Nvoc and MeNvoc, also can be used for method and composition as herein described or uses with method and composition as herein described.Known other protecting group also can be used for method and composition as herein described or uses with method and composition as herein described in this technology.
Only for instance, END CAPPED GROUP/protecting group can be selected from following groups.
Figure A20058004446400441
Other protecting group is to be described in Greene and Wuts, Protective Groups in Organic Synthesis, the third edition, John Wiley ﹠amp; Sons, New York, NY, in 1999, described reference is incorporated herein by reference in full.
In treatment was used, the patient who suffers from disease, illness or deficiency disorder was given in the composition throwing that will contain modified non-natural amino acid polypeptides, presents in an amount at least sufficient to cure or suppress at least to a certain extent the symptom of disease, deficiency disorder or illness.Described amount is defined as " treatment significant quantity ", and it will be decided on the severity of disease, deficiency disorder or illness and the course of disease, previous treatment, patient's healthy state with to the reaction of medicine and treatment doctor's judgement.The those skilled in the art is to determining that by normal experiment (for example, dosage escalation clinical trial) described treatment significant quantity has abundant understanding.
Term " treatment " is to be used to refer to preventative and/or therapeutic treatment.
Non-naturally encoded amino acid polypeptide provided herein can comprise through isotope-labeled compound, and it has one or more for having the atomic mass that is different from common atomic mass of occurring in nature or total mass number or the atom institute metathetical atom of total mass number.Can incorporate the isotropic substance that isotopic example in the The compounds of this invention comprises hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine into, respectively such as being 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl.As herein described some through isotope-labeled compound, for example will such as 3H and 14The radio isotope of C is incorporated those compounds wherein into, can be used for medicine and/or matrix organization's distributional analysis.In addition, use (that is, such as deuterium 2H) the treatment advantage that can provide some to cause owing to higher metabolic stability usually is provided coordination, for example, and the in vivo transformation period of prolongation or the dosage demand of reduction.
All isomer that include, but is not limited to diastereomer, enantiomer and its mixture are regarded as the part of composition as herein described.In addition or among other embodiment, non-naturally encoded amino acid polypeptide gives in throwing and metabolism takes place behind the organism that needs and produce metabolite, and described subsequently metabolite is used for producing the effect of wanting, and comprises the result of treatment of wanting.At other or among the embodiment be the active metabolite of non-natural amino acids coding polypeptide in addition.
In some cases, non-naturally encoded amino acid polypeptide can be the tautomeric forms existence.In addition, non-naturally encoded amino acid polypeptide as herein described can the non-solvent form and is existed with the solvation form that the pharmaceutically acceptable solvent such as water, ethanol or the like forms.The solvation form is regarded as that also announcement is arranged in this article.Those skilled in the art will realize that some compounds herein can some tautomeric forms exist.All described tautomeric forms are regarded as the part of composition as herein described.
Except as otherwise noted, otherwise adopt mass spectroscopy, NMR, HPLC, protein chemistry, biological chemistry, recombinant DNA technology and the pharmacological ordinary method belong to this technical skills scope.
Embodiment
I. foreword
The GH that comprises at least one alpha-non-natural amino acid is provided (for example, hGH) molecule in the present invention.In certain embodiments of the present invention, (for example, hGH) polypeptide comprises at least a posttranslational modification to have the GH of at least one alpha-non-natural amino acid.In one embodiment, described at least a posttranslational modification comprises the molecule that includes, but is not limited to following material that utilizes the known chemical process that is applicable to the specific reactivity group of those skilled in the art will comprise second reactive group and is connected with at least one alpha-non-natural amino acid that comprises first reactive group: mark, dyestuff, polymkeric substance, water-soluble polymers, the derivative of polyoxyethylene glycol, the photocrosslinking agent, radionuclide, cytotoxin compounds, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, the antisense polynucleotide, carbohydrate, water-soluble branch-shape polymer, cyclodextrin, inhibition Yeast Nucleic Acid, biomaterial, nanoparticle, spin labeling, fluorophore, metallic part, radioactive segment, novel functional group, covalently or non-covalentlyly with the group of other interaction of molecules, light cage latching segment (photocaged moiety), but actinic radiation excitation portion, the intramolecular photosensitization part, vitamin H, the derivative of vitamin H, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, the side chain of elongation, sugar through the carbon connection, redox active agent, amino thioic acid sulfoacid, toxic moiety, through isotope-labeled part, the biophysics probe, phosphorescent group, chemiluminescent groups, the intensive group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable mark, small molecules, quantum dot, the nanometer transmitter, the radioactive nuleus thuja acid, the radioactivity transmitter, any combination of neutron capture agent or above-mentioned substance or any other desired compound or material.For example, described first reactive group is that the alkynyl part (includes, but is not limited to when propargyl is also sometimes referred to as acetylene moiety, alpha-non-natural amino acid to the propargyloxy phenylalanine in) and described second reactive group be the azido-part, and utilize [3+2] cycloaddition chemical process.In another embodiment, first reactive group is that the azido-part (include, but is not limited to alpha-non-natural amino acid to azido--L-phenylalanine in) and second reactive group are the alkynyl part.At modified GH of the present invention (for example, hGH) among some embodiment of polypeptide, use comprises at least one alpha-non-natural amino acid (including, but is not limited to contain the alpha-non-natural amino acid of ketone group functional group) of at least a posttranslational modification, and wherein said at least a posttranslational modification comprises the carbohydrate part.In certain embodiments, posttranslational modification is in vivo to carry out in eukaryotic cell or in non-eukaryotic cell.
In certain embodiments, protein comprises at least a posttranslational modification of carrying out by a kind of host cell in vivo, and wherein said posttranslational modification is not generally undertaken by another kind of host cell type.In certain embodiments, protein comprises at least a posttranslational modification of carrying out by eukaryotic cell in vivo, and wherein said posttranslational modification is not generally undertaken by non-eukaryotic cell.The example of posttranslational modification includes, but is not limited to glycosylation, acetylizing, acylation, lipid-modified, palmitoylation effect, cetylate interpolation, phosphorylation, the glycolipid key is modified or the like.In one embodiment, posttranslational modification comprise by GlcNAc-asparagine key oligosaccharides is connected with asparagine (including, but is not limited to wherein, oligosaccharides comprises (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc or the like situation).In another embodiment, posttranslational modification comprises by GalNAc-Serine key, GalNAc-Threonine key, GlcNAc-Serine key or GlcNAc-Threonine key oligosaccharides (including, but is not limited to Gal-GalNAc, Gal-GlcNAc etc.) is connected with Serine or Threonine.In certain embodiments, protein of the present invention or polypeptide can comprise secretion sequence or positioning sequence, Kang Yuan Decision decides disjunction mark label, FLAG label, polyhistidyl label, GST fusions or the like.The example of secretory signal sequence includes, but is not limited to prokaryotic secretion signal sequence, eucaryon secretory signal sequence, 5 ' optimizing with the eucaryon secretory signal sequence that is used for bacterial expression, novel secretory signal sequence, pectate lyase secretory signal sequence, OmpA secretory signal sequence and phage secretory signal sequence.The example of secretory signal sequence includes, but is not limited to STII (protokaryon), Fd GIII and M13 (phage), Bgl2 (yeast) and is derived from the signal sequence bla of transposon.
Protein of being paid close attention to or polypeptide can contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or 10 or 10 above alpha-non-natural amino acids.Described alpha-non-natural amino acid can be identical or different, for example, in protein, can there be the different position more than 1,2,3,4,5,6,7,8,9,10 or 10 that comprises alpha-non-natural amino acids different more than 1,2,3,4,5,6,7,8,9,10 or 10.In certain embodiments, being present in the specific amino acids of at least one (but being less than sum) in the proteinic naturally occurring form replaces through alpha-non-natural amino acid.
The invention provides based on the method and composition that comprises at least one non-naturally encoded amino acid whose GH supergene family member (being in particular hGH).Amino acid that at least one is non-naturally encoded is introduced and can be made among the GH supergene family member and relate to (including, but is not limited to) with one or more non-naturally encoded amino acid whose particular chemical reactions but do not used with the chemical action that combines that 20 seed amino acids of common existence react.In certain embodiments, comprising non-naturally encoded amino acid whose GH supergene family member is connected with water-soluble polymers such as polyoxyethylene glycol (PEG) by non-naturally encoded amino acid whose side chain.The invention provides a kind of high efficiency method with PEG derivatives selectively ground modifying protein, it relates in response to the amino acid of selecting codon with non-genetic coding (including, but is not limited to contain functional group or substituent those amino acid of not existing and including, but is not limited to ketone part, nitrine part or acetylene moiety in 20 kinds of natural amino acid of incorporating into) selectivity and incorporates in the protein and modify those amino acid with reactive suitable substance P EG derivative subsequently.In case after incorporating into, can come the modified amino acid side chain by utilizing the known particular functional group or the substituent chemical process that are present in the non-naturally encoded amino acid of being applicable to of those skilled in the art with that.Multiple known chemical process is applicable to that the present invention incorporates water-soluble polymers in the protein into.Described method include, but is not limited to make respectively include, but is not limited to acetylene or azido derivant take place Hu Shi root [3+2] cycloaddition reaction (referring to, for example, Padwa, A., Comprehensive Organic Synthesis, the 4th volume, (1991), Trost, B.M. compiles, Pergamon, Oxford is in the 1069-1109 page or leaf; And Huisgen, R., 1, 3-Dipolar Cycloaddition Chemistry, (1984), Padwa, A. compiles, Wiley, New York is in the 1-176 page or leaf).
Because Hu Shi root [3+2] cycloaddition method relates to cycloaddition reaction rather than nucleophilic substitution reaction, can modify protein with very high selectivity.Its reaction can be under room temperature, aqueous conditions be added in the reaction mixture by Cu (I) salt with catalytic amount and is carried out with splendid regioselectivity (1,4＞1,5).Referring to, for example, people such as Tomoe, (2002) J. Org.Chem.67:3057-3064; With people such as Rostovtsev, (2002) Angew.Chem.Int. Ed.41:2596-2599; With WO 03/101972.Can comprise having appropriate functional group or substituent almost any molecule by the molecule that [3+2] cycloaddition reaction joins in the protein of the present invention, include, but is not limited to azido-or acetylene-derivative.These molecules can join respectively in non-natural amino acid (including, but is not limited to the propargyloxy phenylalanine) with ethynyl or the non-natural amino acid (including, but is not limited to the triazobenzene L-Ala) with azido-.
Generally irreversible under reducing environment by the five-ring that Hu Shi root [3+2] cycloaddition reaction obtains, and under aqueous environments, in the long-term time, have hydrolytic stability.Therefore, the physics of multiple material and chemical property can be modified under demanding aqueous conditions with active PEG derivative of the present invention.Even what is more important because nitrine part and acetylene moiety be each other specific (and for example not with the amino acid of 20 kinds of common genetic codings in any amino acid react), so can on one or more privileged site, modify with very high selectivity to protein.
The present invention also provides the derivative of the water-soluble and stability to hydrolysis of the tool of PEG derivative with one or more acetylene moiety or nitrine part and relevant hydrophilic polymer.Contain the PEG polymer derivant pair of acetylene moiety and have high selectivity in response to the nitrine part coupling of selecting codon optionally to introduce in the protein.Similarly, the PEG polymer derivant that contains nitrine part pair has high selectivity with the acetylene moiety coupling of optionally introducing in the protein in response to the selection codon.
More particularly, the nitrine part is including (but not limited to) the derivative of alkyl diazoimide part, aryl azide part and these nitrine part.As long as keep the acetylene atopy, the derivative of alkyl diazoimide part and aryl azide part can comprise other substituting group.Acetylene moiety comprises alkyl acetylene moiety and aryl ethane part and derivative separately.As long as keep the nitrine atopy, the derivative of alkyl acetylene moiety and aryl ethane part can comprise other substituting group.
The invention provides have various functional groups, the material of substituting group or part with include, but is not limited to the binding substances of other material of following material: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; The photocrosslinking agent; Radionuclide; Cytotoxin compounds; Medicine; Affinity labelling; Photoaffinity labeling; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; The metal chelating; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; The antisense polynucleotide; Carbohydrate; Water-soluble branch-shape polymer; Cyclodextrin; Inhibition Yeast Nucleic Acid; Biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; Covalently or non-covalentlyly with the group of other interaction of molecules; Light cage latching segment; But actinic radiation excitation portion; The intramolecular photosensitization part; Vitamin H; The derivative of vitamin H; The vitamin H analogue; The part of incorporating heavy atom into; But the group of chemical cracking; But the group of photo-cleavage; The side chain of elongation; Sugar through the carbon connection; Redox active agent; Amino thioic acid sulfoacid; Toxic moiety; Through isotope-labeled part; The biophysics probe; Phosphorescent group; Chemiluminescent groups; The intensive group of electronics; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable mark; Small molecules; Quantum dot; The nanometer transmitter; The radioactive nuleus thuja acid; The radioactivity transmitter; Neutron capture agent; Or any combination of above-mentioned substance, or any other wanted compound or material.The present invention also comprises the binding substances of material with nitrine part or acetylene moiety and the PEG polymer derivant with corresponding acetylene moiety or nitrine part.For instance, contain nitrine PEG polymkeric substance partly can contain the non-genetic coding with acetylene functional group in protein amino acid whose position and bioactive molecules coupling mutually.PEG and the bioactive molecules keyed jointing of coupling mutually include, but is not limited to Hu Shi root [3+2] cycloaddition keyed jointing.
Fully determine in this technology PEG can be used for the surface of modified biological material (referring to, for example, United States Patent (USP) the 6th, 610, No. 281; Mehvar, R., J.Pharm Pharm Sci., 3 (1): 125-136 (2000), it is incorporated herein by reference).The present invention comprises that also to comprise surface with one or more reactive nitrine position or acetylene position of the present invention by the biomaterial of Hu Shi root [3+2] cycloaddition keyed jointing with the polymkeric substance that contains nitrine part or acetylene moiety of described surperficial coupling mutually with one or more.Biomaterial and other material also can be by being different from nitrine keyed jointing or acetylene keyed jointing keyed jointing (such as, by comprising the keyed jointing of carboxylic acid, amine, alcohol or thiol moiety) with nitrine or acetylene activatory polymer derivant mutually coupling so that nitrine partly or acetylene moiety can be used for afterreaction.
The present invention includes a kind of synthetic method that contains the nitrine part and contain the polymkeric substance of acetylene moiety of the present invention.Under the situation of the PEG derivative that contains nitrine part, the nitrine part can be directly and the carbon atom of polymkeric substance bond mutually.Perhaps, the PEG derivative that contains nitrine part can prepare so that make the polymkeric substance of gained have the nitrine part at its end by making the keyed jointing agent that has a nitrine part at an end link to each other with the activated polymer of routine to fetch.Under the situation of the PEG derivative that contains acetylene moiety, acetylene moiety can be directly and the carbon atom of polymkeric substance bond mutually.Perhaps, the PEG derivative that contains acetylene moiety can fetch preparation so that make the polymkeric substance of gained have acetylene moiety at its end by the keyed jointing agent that has an acetylene moiety at an end is linked to each other with the activated polymer of routine.
More particularly, under the situation of the PEG derivative that contains the nitrine part, water-soluble polymers with at least one activity hydroxy part react and produce have thereon have more reactive part (such as, mesylate, trifluoro esilate, tosylate or halogen leavings group) be substituted polymkeric substance.The preparation and the use that contain the PEG derivative of alkylsulfonyl acid halogenide, halogen atom and other leavings group are known to those of ordinary skill in the art.The polymkeric substance that is substituted of gained then reacts and partly replaces described reactive part that has more at the end of polymkeric substance with nitrine.Perhaps, have the water-soluble polymers of at least one active nucleophilic or electrophilic part with the keyed jointing agent reaction that has nitrine part at an end in case between PEG polymkeric substance and keyed jointing agent formation covalent linkage and make nitrine partly be positioned at the end of polymkeric substance.The nucleophilic and the electrophilic part that comprise amine, mercaptan, hydrazides, hydrazine, alcohol, carboxylicesters, aldehyde, ketone, thioesters or the like are known to those of ordinary skill in the art.
More particularly, containing under the PEG derivative situation of acetylene moiety, the water-soluble polymers with at least one activity hydroxy part reacts and by the precursor displacement halogen or other activatory leavings group that contain acetylene moiety.Perhaps, have the water-soluble polymers of at least one active nucleophilic or electrophilic part with the keyed jointing agent reaction that has acetylene moiety at an end in case between PEG polymkeric substance and keyed jointing agent formation covalent linkage and make acetylene moiety be positioned at the end of polymkeric substance.The use of halogen part, activatory leavings group, nucleophilic and electrophilic part is fully definite for the those skilled in the art in organic synthesis part with in the preparation of PEG derivative and in using.
The present invention also provides a kind of protein is carried out selective modification so that other material (include, but is not limited to water-soluble polymers, such as, PEG and PEG derivative, it contains nitrine part and acetylene moiety) is joined the method in the modified protein.The PEG derivative that contains nitrine part and acetylene moiety can be used for changing biocompatibility, stability, solvability and the surface of shortage immunogenicity outbalance and the character of molecule, and provides the means than previously known in this technology that the PEG derivative is connected with protein to have more optionally means simultaneously.
II. tethelin supergene family
Following protein comprises by those protein of the coded by said gene of tethelin (GH) supergene family (Bazan, F., Immunology Today 11:350-354 (1990); Bazan, J.F.Science 257:410-413 (1992); Mott, H.R. and Campbell, I.D., Current Opinion in Structural Biology 5:114-121 (1995); Silvennoinen, O. and Ihle, J.N., SIGNALLING BY THE HEMATOPOIETIC CYTOKINERECEPTORS (1996)): tethelin, prolactin, the placenta lactogen, erythropoietin (EPO), thrombosis element (TPO), IL-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), interferon-alpha, interferon-, the ε Interferon, rabbit, IFN-, omega interferon, the τ Interferon, rabbit, granulocyte colony-stimulating factor (G-CSF), rHuGM-CSF (GM-CSF), macrophage colony stimulating factor (M-CSF) and myocardial nutrition element-1 (CT-1) (" GH supergene family ").Can expect that the other member of this gene family will differentiate by gene clone and sequencing in the future.Although the member of GH supergene family generally has limited amino acid or dna sequence dna identity, it has similar secondary and tertiary structure.Total constitutional features is differentiated the newcomer of gene family easily and non-natural method of amino-acids as herein described and composition is applied similarly.Consider the structural homology degree between the member of GH supergene family, can utilize the present invention that non-naturally encoded amino acid is incorporated among any member of GH supergene family.Each member in this protein families all comprises a four-helix bundle, shows its general structure in Fig. 1.The general structure that in Fig. 2, Fig. 3, Fig. 4 and Fig. 5, shows family member hGH, EPO, IFN α-2 and G-CSF respectively.
Comprise G-CSF (people such as Zink, FEBS Lett.314:435 (1992); People such as Zink, Biochemistry 33:8453 (1994); People such as Hill, Proc.Natl.Acad.Sci.USA 90:5167 (1993)), GM-CSF (Diederichs, people such as K., Science 154:1779-1782 (1991); People such as Walter, J. Mol.Biol 224:1075-1085 (1992)), IL-2 (Bazan, J.F. and McKay, D.B., Science 257:410-413 (1992)), IL-4 (people such as Redfield, Biochemistry 30:11029-11035 (1991); People such as Powers, Science 256:1673-1677 (1992)) and IL-5 (people such as Milburn, Nature 363:172-176 (1993)) although structure of a large amount of cytokines is determined by X-ray diffraction and NMR research and is lacked significant primary sequence homology, and it shows surprising conservative property with regard to the GH structure.Based on modeling and other research, IFN is considered to member (people such as Lee, the J.Interferon Cytokine Res.15:341 (1995) of this family; People such as Murgolo, Proteins 17:62 (1993); People such as Radhakrishnan, Structure 4:1453 (1996); People such as Klaus, J.Mol.Biol.274:661 (1997)).Based on modeling and mutation research, EPO is considered to member (people such as Boissel, the J. Biol.Chem.268:15983-15993 (1993) of this family; People such as Wen, J.Biol.Chem.269:22839-22846 (1994)).Think that now all above-mentioned cytokines and somatomedin constitute a big gene family.
Except that having similar secondary and tertiary structure, the character that the member of this family has must make cell surface receptor generation oligomerization with signaling path in the activating cells for it.Some GH family members that include, but is not limited to GH and EPO combine with the acceptor of single type and make it form homodimer.Other family member who includes, but is not limited to IL-2, IL-4 and IL-6 with combine more than one type acceptor and make described acceptor form heterodimer or high grade collecting body (people such as Davis, (1993), Science 260:1805-1808; People such as Paonessa, (1995), EMBO is J.14:1942-1951; Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995)).Mutation research shows, these other cytokine and somatomedin contains a plurality of receptor binding sites as GH, be generally 2 receptor binding sites, and in regular turn in conjunction with its homoreceptor (Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995); People such as Matthews, (1996) Proc.Natl.Acad.Sci.USA 93:9471-9476).As GH, these other family member's principal recipient combining site mainly is present in 4 α spirals and the A-B ring.Specific amino acids in the helical bundle of participation receptors bind is inequality between the kinsfolk.Structurally be relevant and constitute second big multigene family with the interactional most of cell surface receptor of the member of GH supergene family.Referring to, for example, United States Patent (USP) the 6th, 608, No. 183, it is incorporated herein by reference.
The common conclusions that obtains from each member's of GH supergene family mutation research is that the ring that connects the α spiral does not generally trend towards relating in the receptors bind.Particularly, lack B-C ring manifest for the family member's of most (if not all words) the receptors bind for nonessential.For this reason, in the member of GH supergene family, the B-C ring can be through non-naturally encoded aminoacid replacement as described herein.The aminoacid replacement that A-B ring, C-D ring (with the Interferon, rabbit/IL-10 sample member's of GH superfamily D-E ring) also can exist through non-natural.Do not trend towards relating in the receptors bind yet and can be to be used to introduce the amino acid whose position that non-natural exists yet near spirane structure A and away from the amino acid of last spirane structure.In certain embodiments, any position in including, but is not limited to preceding 1,2,3,4,5,6 of A-B, B-C, C-D or D-E ring, amino acid whose ring structure more than 7 or 7 is with non-naturally encoded aminoacid replacement.In certain embodiments, in A-B, B-C, C-D or D-E ring back 1,2,3,4,5,6, amino acid more than 7 or 7 with one or more non-naturally encoded aminoacid replacement.
Some member who includes, but is not limited to the GH family of EPO, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12 p35, IL-13, IL-15 and interferon-is contained the sugar that N-is connected and/or O-connects.Glycosylation position in the protein almost completely be present in the ring zone in and be not present in the α helical bundle.Because ring zone does not generally relate in the receptors bind and because it is the covalently bound position that is used for glycosyl group, so its useful position that can be the aminoacid replacement that is used for that non-natural is existed introduce protein.Comprise the position that amino acid that N-connects the glycosylation position is connected with O-can the naturally occurring aminoacid replacement of right and wrong in protein, reason is that these amino acid are surperficial exposures.Therefore, native protein mass-energy allows can have huge glycosyl group be connected with protein at these positions and the glycosylation position trends towards being positioned at away from the receptor binding site place.
Might be other member who finds in the future the GH supergene family.The newcomer of GH supergene family can be by to the area of computer aided secondary of predicted protein matter sequence and tertiary structure analysis and by being used for through design differentiating that the selection technology of the molecule that combines with particular target differentiates.The member of GH supergene family has 4 or 5 amphiphilic spirane structures that connect by non-helical shape amino acid (ring zone) usually.Protein can contain the hydrophobicity signal sequence to promote emiocytosis at its N end.The member of the GH supergene family that these were found afterwards is also included among the present invention.Related application is to be disclosed as the international application of the title of WO 05/074650 for " Modified Four Helical BundlePolypeptides and Their Uses " on August 18th, 2005, and it incorporates this paper by reference into.
Therefore, providing the description of tethelin supergene family, only is for illustration purposes and only to be example, and is not to be the restriction to the scope of method as herein described, composition, strategy and technology.In addition, mention that in the application's case the GH polypeptide is intended to use the example of this generic term as any GH supergene family member.Therefore, should be appreciated that herein any member that can similarly be applicable to the GH supergene family about hGH polypeptide or the described modification of protein and chemical action, comprise specific those members that list herein.
III. the general recombinant nucleic acid method of using for the present invention
In many embodiment of the present invention, will use recombination method to separate, clone and usually be to be used for changing the GH that coding pays close attention to (for example, the hGH) nucleic acid of polypeptide.Described embodiment is used for (including, but is not limited to) protein expression or (for example, hGH) uses described embodiment during the generation of the varient of polypeptide, derivative, expression cassette or other sequence being derived from GH.In certain embodiments, the sequence with coding polypeptide of the present invention operably is connected to allogeneic promoter.The generation of the separation of hGH and GH is United States Patent (USP) for example the 4th, 601, No. 980, the 4th, 604, No. 359, the 4th in the host cell, 634, No. 677, the 4th, 658, No. 021, the 4th, 898, No. 830, the 5th, 424, No. 199, the 5th, 795, No. 745, the 5th, 854, No. 026, the 5th, 849, No. 535, the 6th, 004, No. 931, the 6th, 022, No. 711, the 6th, 143, No. 523 and the 6th, 608, to be described in No. 183, described patent is incorporated herein by reference.
The aminoacid sequence that the nucleotide sequence that coding comprises non-naturally encoded amino acid whose hGH polypeptide can be based on the parent polypeptide that includes, but is not limited to have the aminoacid sequence shown in the SEQ ID NO:2 (hGH) comes synthetic, and then change nucleotide sequence so as the introducing that realizes the related amino acid residue (promptly, incorporate into or replace) or remove (that is, disappearance or replace).Nucleotide sequence can be modified by rite-directed mutagenesis expediently according to conventional methods.Perhaps, nucleotide sequence can prepare by chemosynthesis, includes, but is not limited to by using oligonucleotide synthesizer (wherein oligonucleotide is based on the amino acid sequence of polypeptide design of wanting) and preferentially being chosen in those favourable codons in the host cell of generation recombinant polypeptide be prepared.For example, some encoding section branch are wanted the small oligonucleotide of polypeptide or to connect chain reaction by PCR, connection to synthesize and assemble.Referring to, for example, people such as Barany, Proc.Natl.Acad.Sci.88:189-193 (1991); United States Patent (USP) the 6th, 521, No. 427, it is incorporated herein by reference.
The present invention utilizes the routine techniques in the genetic recombination field.The basic article that announcement is used for general method of the present invention comprises people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition, 2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocols inMolecular Biology (people such as Ausubel compiles, 1994).
The general article of describing Protocols in Molecular Biology comprises Berger and Kimmel, Guide to Molecular Cloning Techniques.Methods in Enzymology, the 152nd volume,Academic Press, Inc., San Diego, CA (Berger); People such as Sambrook, Molecular Cloning-A Laboratory Manual (the 2nd edition), 1-3 Volume,Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") and Current Protocols in Molecular Biology, people such as F.M.Ausubel compile, Current Protocols, a jointventure between Greene Publishing Associates, Inc.and John Wiley ﹠amp; Sons, Inc. (being replenished) (" Ausubel ") via 1999.Sudden change described in these articles, the use of carrier, promotor and many other related subjects about the generation of (including, but is not limited to) gene or polynucleotide, described gene or polynucleotide comprise and are used to produce proteinic selection codon, quadrature tRNA, orthogonal synthetic enzyme and its pairing that comprises alpha-non-natural amino acid.
Use various types of sudden changes to be used for various purposes among the present invention, include, but is not limited to produce novel synthetic enzyme or tRNA, make the sudden change of tRNA molecule, make the polymerized nucleoside acid mutation of coding synthetic enzyme, produce the tRNA storehouse, produce the synthetic enzyme storehouse, produce and select codon, the selection codon of coding alpha-non-natural amino acid is inserted in the protein of being paid close attention to or polypeptide.Described sudden change includes, but is not limited to the dna mutation that rite-directed mutagenesis, random point mutation, homologous recombination, DNA reorganization or other return sudden change that mutation method, chimeric construct, use contain the template of uridylic, oligonucleotide orthomutation, modify through thiophosphatephosphorothioate, sudden change of using the breach double-stranded DNA or the like or its any combination.Other appropriate method comprises a mispairing reparation, the sudden change, restriction-selection and the restriction-purifying that use the rectification of defects host strain, deletion mutantion, repairs or the like by total gene synthetic sudden change, double-strand break.The sudden change that includes, but is not limited to relate to chimeric construct is also included among the present invention.In one embodiment, sudden change can or change or the Given information of the naturally occurring molecule that suddenlys change is instructed by naturally occurring molecule, and described information comprises (but being not limited to) sequence, sequence comparison, physical properties, secondary, three grades or quaternary structure, crystalline structure or the like information.
Being seen herein article and these programs of case description.Out of Memory see the following discloses case and the reference wherein quoted in: people such as Ling, Approaches to DNA mutagenesis:an overview, Anal Biochem.254 (2): 157-178 (1997); People such as Dale, 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 ﹠amp; 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 ﹠amp; Molecular Biology(D.MJ. compiles for Eckstein, F. and Lilley, and Springer Verlag is Berlin) in (1987); Kunkel, Rapid and efficient site-specific mutagenesiswithoutphenotypic selection, Proc.Natl.Acad.Sci.USA82:488-492 (1985); People such as Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol.154,367-382 (1987); People such as Bass, Mutant Trp repressors with new DNA-bindingspecificities, Science242:240-245 (1988); Zoller ﹠amp; Smith, Oligonucleotide-directedmutagenesis using M13-derived vectors:an efficient and general procedure for theproduction of point mutations in any DNA fragment Nucleic Acids Res.10:6487-6500 (1982); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis of DNA fragments clonedinto M13 vectors, Methods in Enzymol.100:468-500 (1983); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis:a simple method using two oligonucleotide primersand a single-stranded DNA template, Methods in Enzymol.154:329-350 (1987); People such as Taylor, The use of phosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl.Acids Res.13:8749-8764 (1985); People such as Taylor, The rapid generation ofoligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res.13:8765-8785 (1985); Nakamaye ﹠amp; Eckstein, Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenests, Nucl.Acids Res.14:9679-9698 (1986); People such as Sayers, 5 '-3 ' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. Acids Res.16:791-802 (1988); People such as Sayers, Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restriction endonucleases in the presenceof ethidium bromide, (1988) Nucl.Acids Res.16:803-814; People such as Kramer, The gapped duplexDNA approach to oligonucleotide-directed mutation construction, Nucl. Acids Res.12:9441-9456 (1984); Kramer ﹠amp; Fritz Oligonucleotide-directed construction of mutations viagapped duplex DNA, Methods in Enzymol.154:350-367 (1987); People such as Kramer, Improvedenzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directedconstruction of mutations, Nucl.Acids Res.16:7207 (1988); People such as Fritz, Oligonucleotide-directed construction of mutations:a gapped duplex DNA procedure withoutenzymatic reactions in vitro, Nucl.Acids Res.16:6987-6999 (1988); People such as Kramer, Difierentbase/base mismatches are corrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E.coli, Cell38:879-887 (1984); People such as Carter, Improvedoligonucleotide site-directed mutagenesis using M13 vectors, Nucl.Acids Res.13:4431-4443 (1985); Carter, Improved oligonucleotide-directed mutagenesis using Ml3 vectors, Methods in Enzymol.154:382-403 (1987); Eghtedarzadeh ﹠amp; Henikoff, Use ofoligonucleotides to generate large deletions, Nucl.Acids Res.14:5115 (1986); People such as Wells, Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil. Trans.R.Soc.Lond.A 317:415-423 (1986); People such as Nambiar, Total synthesis and cloning ofa gene coding for the ribonuclease Sprotein, Science223:1299-1301 (1984); Sakmar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin), Nucl.Acids Res.14:6361-6372 (1988); People such as Wells, Cassette mutagenesis:an efficient method for generation of multiplemutations at defined sites, Gene34:315-323 (1985); People such as Grundstr  m, Oligonucleotide-directed mutagenesis by microscale ' shot-gun ' gene synthesis, Nucl.Acids Res.13:3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand break repairin plasmids of Escherichia coli:amethod for site-specific mutagenesis, P Roc.Natl.Acad.Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology4:450-455 (1993); People such as Sieber, 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).Other detailed description of relevant many aforesaid methods is found in Methods in EnzymologyIn the 154th volume, it also describes the effective control to the troubleshooting queueing problem of using various mutation methods.
(for example) oligonucleotide of sudden change of the present invention that is used for for example making the sudden change of synthetic enzyme storehouse or changes tRNA is normally according to Beaucage and Caruthers, Tetrahedron Letts.22 (20): 1859-1862, (1981) described solid phase phosphoramidite three ester methods, as people such as Needham-VanDevanter, Nucleic Acids Res., (for example) described in the 12:6159-6168 (1984) uses automated synthesizer and chemosynthesis.
The present invention also relates to by quadrature tRNA/RS the eukaryotic host cell of in vivo alpha-non-natural amino acid being incorporated into, non-eukaryotic host cell and organism.Host cell is with polynucleotide of the present invention or comprises construction (include, but is not limited to carrier of the present invention, it can for example be cloning vector or the expression vector) genetic modification (including, but is not limited to conversion, transduction or transfection) in addition of polynucleotide of the present invention.For example, with quadrature tRNA, quadrature tRNA synthetic enzyme with treat that the proteinic coding region of derivatize operably is connected to the genetic expression controlling elements that works in the principal host of institute cell.Carrier can (for example) be plasmid, cement grain, phage, bacterium, virus, exposed polynucleotide or bonded polynucleotide form.Carrier is to introduce in cell and/or the microorganism by the standard method that comprises following method: electroporation (people such as Fromm, Proc.Natl.Acad.Sci.USA82,5824 (1985)), by viral vector infection, in the matrix of globule or particle or have from the teeth outwards the small-particle of nucleic acid high speed trajectory osmosis (people such as Klein, Nature327.70-73 (1987)) and/or like that.
Can cultivate in conventional nutritional medium through the host cell of transforming, described nutritional medium is in due course for such as screening step, activation promotor or select the activity of transformant and upgrading in addition.Can optionally these cells be cultivated in transgenic organism.Include, but is not limited to comprise Freshney (1994) about other useful reference of cellular segregation and cultivation (for example, about subsequently separate nucleic acid) Culture of Animal Cells, a Manual of Basic Technique, the 3rd edition, Wiley-Liss, New York and the reference of wherein being quoted; People such as Payne (1992) Plant Cell and Tissue Culture in Liquid SystemsJohn Wiley ﹠amp; Sons, Inc.New York, NY; Gamborg and Phillips (volume) (1995) Plant Cell, Tissue and Organ CultureFundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (volume) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, FL.
Some methods of knowing that target nucleic acid is introduced in the cell are available, and any method wherein can be used among the present invention.These methods comprise: make recipient's cell and the fusion of bacterium protoplastis, the electroporation that contain DNA, projectile body bombardment and infect (hereinafter further discussing) etc. with virus vector.Bacterial cell can be used to increase the quantity of the plasmid that contains DNA construction of the present invention.Make bacterial growth to the plasmid in logarithmic phase and the bacterium can separate by known the whole bag of tricks in this technology (referring to, for example, Sambrook).In addition, be used for from the test kit of bacterium plasmid purification be commercially available (referring to, for example, all from the EasyPrep of Pharmacia Biotech TM, FlexiPrep TMStrataClean from Stratagene TMWith QIAprep from Qiagen TM).Then to through separating and the plasmid of purifying is further handled with generation and is used for transfectional cell or incorporate in the related vector other plasmid with the infection biological body into.Typical carrier contains transcribes with translation termination, transcribes and translation initiation sequence and promotor (can be used for regulating the particular target expression of nucleic acids).Carrier optionally comprises expression casette, described expression cassette contain at least one independently the terminator sequence, allow the sequence (including, but is not limited to shuttle vectors) that box duplicates and be suitable for prokaryotic system and the selectable marker of eukaryotic system in eukaryote or prokaryotic organism or two kinds of biology.Carrier is suitable for duplicating and integrating in prokaryotic organism, eukaryote or two kinds of biologies.Referring to, Gillam ﹠amp; Smith, Gene8:81 (1979); People such as Roberts, Nature, 328:731 (1987); Schneider, people such as E., Protein Expr. Purif.6 (1): 10-14 (1995); Ausubel, Sambrook, Berger (all are the same).Bacterium that can be used for cloning and the catalogue of phage (for example) are provided by ATCC, are for example published by ATCC The ATCC Catalogue of Bacteria and Bacteriophage(1992) people's (volume) such as Ghema.Also see people (1992) such as Watson about other base program of sequencing, clone and molecular biological others and the theoretical point view on basis Recombinant DNA (the 2nd edition)Scientific American Books is among the NY.In addition, basically any nucleic acid is (with any nucleic acid through mark almost, no matter be also criteria of right and wrong thing of standard substance) can be conventional thing or the standard substance that order in any source from various commercial source, described commercial source is such as Midland Certified Reagent Company (Midland, TX can be at www. Mcrc.comLast acquisition), (Ramona, CA can be at www. for The Great American Gene Company Genco.comLast acquisition), (Chicago, IL can be at www. for ExpressGen Inc. Expressgen.comLast acquisition), (Alameda is CA) with many other commercial source for Operon Technologies Inc..
(a) select codon
The genetic code subframe of selection codon expansion protein biosynthesizing machine of the present invention.For example, select codon to include, but is not limited to single three base codons, nonsense codon (such as the terminator codon that includes, but is not limited to amber codon (UAG), ocher codon or opal codon (UGA)), non-natural codon, 4 or 4 above base codons, rare codon or the like.The those skilled in the art understands easily, can be introduced into the quantitative range broad of the selection codon in the gene of wanting or the polynucleotide, include, but is not limited in the single polynucleotide that is encoding to small part hGH polypeptide, exist more than 1 or 1, more than 2 or 2, more than 3 or 3,4,5,6,7,8,9, the selection codon more than 10 or 10.
In one embodiment, method relates to using selects codon, and it is the terminator codon that is used in vivo incorporating at cell one or more alpha-non-natural amino acids.For example, produce the O-tRNA that identification includes, but is not limited to the terminator codon of UAG, and make it that amino acidylate take place with desired alpha-non-natural amino acid by O-RS.Naturally occurring host's aminoacyl-tRNA synthetase can not be discerned this O-tRNA.The terminator codon that includes, but is not limited to TAG is introduced at the concern position that conventional rite-directed mutagenesis can be used in the polypeptide of being paid close attention to.Referring to, for example, Sayers, people such as J.R. (1988), 5 '-3 ' Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res,16:791-802.The nucleic acid of the polypeptide of paying close attention to when O-RS, O-tRNA and coding in vivo in conjunction with the time, alpha-non-natural amino acid is in response to the UAG codon and is incorporated into to obtain containing in specified location the polypeptide of alpha-non-natural amino acid.
In vivo incorporating into of alpha-non-natural amino acid can be finished under the situation of significantly not disturbing eukaryotic host cell.For example, because the inhibition effect of UAG codon decide on the competition between the O-tRNA that includes, but is not limited to amber and suppress tRNA and the eucaryon releasing hormone (including, but is not limited to eRF) (it combines with terminator codon and the peptide that causes in the growth discharges from rrna), so the inhibition effect can be regulated by the expression level of (including, but is not limited to) increase O-tRNA and/or inhibition tRNA.
Alpha-non-natural amino acid also can be encoded by rare codon.For example, when the arginine concentration in vitro protein synthesis reacts reduced, verified rare arginine codon AGG was effective in by the synthetic tRNA through the L-Ala acidylate and inserts Ala.Referring to, for example, people such as Ma, Biochemistry, 32:7939 (1993).In such cases, synthesize tRNA and the naturally occurring tRNAArg competition that is present in as less material in the bacillus coli.Some organisms are not used all triplet codons.Unspecified codon AGA in the micrococcus luteus (Micrococcus luteus) has been used in vitro transcribe/translate insert amino acid in the extract.Referring to, for example, Kowal and Oliver, Nucl.Acid.Res.,25:4685 (1997).Can produce component of the present invention to use these rare codons in vivo.
Select codon also to comprise expansion cipher, include, but is not limited to 4 or 4 above base codons, such as 4,5,6 or 6 above base codons.The example of four base codons includes, but is not limited to AGGA, CUAG, UAGA, CCCU or the like.The example of five base codons includes, but is not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC or the like.Feature of the present invention comprises that expansion cipher is used in inhibition based on frameshit.4 or 4 above base codons can make and include, but is not limited to one or more alpha-non-natural amino acids and insert in same protein.For example, exist down at the sudden change O-tRNA (including, but is not limited to specific frameshift suppressor tRNA) with anticodon loop (for example, having the anticodon loop of 8-10 Nucleotide at least), 4 or 4 above base codons can be read as single amino acids.In other embodiments, the anticodon loop decodable code includes, but is not limited at least four base codons, at least five base codons or hexabasic at least basic codon or at least six above base codons.Because there are 256 kinds of four possible base codons, so can in same cell, use 4 or 4 above base codons a plurality of alpha-non-natural amino acids of encoding.Referring to, people such as Anderson, (2002) Exploring the Limitsof Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of " Shifty " Four-base Codons with a Library Approach in Escherichia coli J. Mol.Biol.307:755-769.
For example, use in vitro biosynthetic means, 4-base codon has been used to alpha-non-natural amino acid is incorporated in the protein.Referring to, for example, people such as Ma, (1993) Biochemistry, 32:7939; With people such as Hohsaka, (1999) J.Am.Chem.Soc,121:34.Use CGGG and AGGU simultaneously the NBD derivative of 2-naphthyl L-Ala and Methionin in vitro to be incorporated in the streptavidin by two frameshift suppressor tRNAs through chemical acylation.Referring to, for example, people such as Hohsaka, (1999) J.Am.Chem.Soc, 121:12194.In vivo in the research, people such as Moore have studied the ability that the tRNALeu derivative with NCUA anticodon suppresses UAGN codon (N can be U, A, G or C), and find that tetrad UAGA can decode rare decoding in 0 or-1 framework simultaneously by the tRNALeu with UCUA anticodon with 13 to 26% efficient.Referring to, people such as Moore, (2000) J.Mol.Biol.,298:195.In one embodiment, can be used among the present invention based on expansion cipher of rare codon or nonsense codon, it can reduce missense at other unwanted position and read with frameshit and suppress.
Concerning specified system, select codon also can comprise a kind of in the natural three base codons, wherein the endogenous system does not use (or seldom using) natural base codon.For example, described system comprise shortage can identify natural three base codons tRNA system and/or wherein three base codons are systems of rare codon.
Select codon optionally to comprise the non-natural base pair.These non-natural base pairs further expand existing genetic alphabet table.An extra base pair makes the number of triplet codon be increased to 125 from 64.The character of the 3rd base pair comprises stable and optionally base pairing, by polysaccharase with high frequency high fidelity effectively enzymatic incorporate among the DNA and effective primer extension continuously in the synthetic back of new life's non-natural base pair.The description that can be suitable for the non-natural base pair of method and composition comprises people such as (for example) Hirao, (2002) An unnatural base pair for incorporating aminoacid analogues intoprotein, Nature Biotechnology, 20:177-182.Also referring to Wu, people such as Y., (2002) J.Am.Chem.Soc.124:14626-14630.Other relevant open case is listed in hereinafter.
Concerning in vivo using, non-natural nucleoside is that film is permeable and formed corresponding triphosphate by phosphorylation.In addition, the genetic information of increase is stable and can not be destroyed by cellular enzymes.Benner and other people attempt utilizing the hydrogen bonding pattern of those patterns that are different from typical Wo Sen-Ke Like (Watson-Crick) centering previously, and wherein the most noticeable example is that iso-C:iso-G is right.Referring to, for example, people such as Switzer, (1989) J.Am.Chem.Soc, 111:8322; With people such as Piccirilli, (1990) Nature,343:33; Kool, (2000) Curr.Opin.Chem.Biol., 4:602.These bases generally speaking to a certain extent with natural base mispairing and can not duplicate enzymatic.Kool and colleague's proof, the hydrophobicity accumulative facies interaction energy between the base replaces hydrogen bonding to impel the formation of base pair.Referring to, Kool, (2000) Curr.Opin.Chem.Biol.,4:602; With Guckian and Kool, (1998) Angew.Chem.Int. Ed.Engl., 36,2825.In the process of being devoted to develop the non-natural base pair that satisfies all above-mentioned requirements, Schultz, Romesberg and colleague have systematically synthesized a series of non-natural hydrophobicity bases and it have been done research.Find that PICS:PICS self pairing is more stable than natural base pair, and can be effectively Klenow fragment by the bacillus coli dna polymerase i (Klenow fragment KF) incorporates among the DNA.Referring to, for example, people such as McMinn, (1999) J.Am.Chem.Soc,121:11585-6; With people such as Ogawa, (2000) J.Am.Chem.Soc, 122:3274.3MN:3MN self pairing energy synthesizes with the efficient and the selectivity that are enough to reach biological function by KF.Referring to, for example, people such as Ogawa, (2000) J.Am.Chem.Soc,122:8803.Yet two bases are served as the chain terminator that further duplicates.Recently, developed and can be used to duplicate PICS self paired mutation DNA polymerase.In addition, reproducible 7AI self pairing.Referring to, for example, people such as Tae, (2001) J.Am.Chem.Soc,123:7439.Also developed novel metal base pairing Dipic:Py, it is forming stable pairing in conjunction with copper (II) back.Referring to, people such as Meggers, (2000) J.Am.Chem.Soc,122:10714.Because expansion cipher and non-natural codon in essence with natural codon quadrature, so method of the present invention can utilize this character to produce its quadrature tRNA.
The translation bypath system also can be used to alpha-non-natural amino acid incorporate into want in the polypeptide.In the translation bypath system, big sequence is merged in the gene but is not translated into protein.Sequence contains to serve as induces rrna to skip sequence and recovers the structure of the prompting of translation in the downstream of inset.
In certain embodiments, protein of being paid close attention in method of the present invention and/or the composition or polypeptide (or its part) are by nucleic acid encoding.Usually, nucleic acid comprises at least 1 selection codon, at least 2 selection codons, at least 3 selection codons, at least 4 selection codons, at least 5 selection codons, at least 6 selection codons, at least 7 selection codons, at least 8 selection codons, at least 9 selection codons, selects codon more than 10 or 10.
The gene of the protein paid close attention to of coding or polypeptide can use the those skilled in the art to know and the method for description herein and undergo mutation that one or more are used to incorporate into the selection codon of alpha-non-natural amino acid to comprise (for example).For example, make the proteinic nucleic acid mutation of being paid close attention to and comprise one or more selection codons that are provided with incorporating into one or more alpha-non-natural amino acids.The present invention includes any described varient, it includes, but is not limited to mutant, and (for example) comprises any protein form of at least one alpha-non-natural amino acid.Similarly, the present invention also comprises corresponding nucleic acids,, has any nucleic acid of one or more selection codons of one or more alpha-non-natural amino acids of coding that is.
The nucleic acid molecule of the protein (such as the hGH polypeptide) that coding pays close attention to is undergone mutation introduce halfcystine with any position of being wanted at polypeptide.Halfcystine is widely used for reactive molecule, water-soluble polymers, protein or various other molecule are incorporated on the protein of being paid close attention to.Be applicable to halfcystine incorporate into polypeptide to want the method in the position be known to those of ordinary skill in the art, such as being described in the United States Patent (USP) the 6th that is incorporated herein by reference, those methods in 608, No. 183 and standard mutating technology.
IV. non-naturally encoded amino acid
There is miscellaneous non-naturally encoded amino acid to be suitable among the present invention.Any amount of non-naturally encoded amino acid can be introduced GH (for example, hGH) in the polypeptide.In general, with respect to 20 kinds of amino acid common, genetic coding (promptly, L-Ala, arginine, asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan), the non-naturally encoded amino acid of introducing is substantially chemically inert.In certain embodiments, non-naturally encoded amino acid comprises and can react to form the side chain functionalities of stable binding substances with non-existent functional group in 20 kinds of common amino acids (including, but is not limited to azido-, ketone group, aldehyde radical and amino oxygen base) effectively and optionally.For example, because optionally reacting, nitrine functional group and alkynes functional group form Hu Shi root [3+2] cycloaddition product, so comprise that (for example, hGH) polypeptide can form stable binding substances with the polymkeric substance that contains alkynyl moiety (including, but is not limited to gather (ethylene glycol)) or the reaction of second polypeptide for the non-naturally encoded amino acid whose GH that contains azido-functional group.
The general structure of a-amino acid is described as follows (formula I).
Figure A20058004446400611
Non-naturally encoded amino acid is generally any structure with top listed formula, and wherein the R group is to be different from used substituent any substituting group in 20 kinds of natural amino acids, and goes among the present invention.Because non-naturally encoded amino acid of the present invention only is different from natural amino acid usually on the structure of side chain, so non-naturally encoded amino acid forms amido linkage with including, but is not limited to natural or non-naturally encoded amino acid whose other amino acid, described amido linkage is to form in the mode identical with its mode that forms in naturally occurring polypeptide.Yet non-naturally encoded amino acid has the side-chain radical that itself and natural amino acid are differentiated.For example, R optionally comprise alkyl-, aryl-, acyl group-, ketone group-, azido--, hydroxyl-, diazanyl, cyano group-, halogen-, hydrazide group, thiazolinyl, alkynyl, ether, thiol group, seleno-, alkylsulfonyl-, boronate, tenth of the twelve Earthly Branches friend's acidic group, phosphate, phosphono, phosphino-, heterocyclic radical, ketenes base, imido grpup, aldehyde radical, ester group, sulfo-acidic group, azanol base, amino or the like or its any combination.But include, but is not limited to comprise the amino acid of the linking agent of photoactivation applicable to the amino acid that the non-natural that other is paid close attention to of the present invention exists, amino acid through spin labeling, fluorescigenic amino acid, the amino acid of bond, metallic amino acid, radioactivity amino acid, amino acid with novel functional group, covalently or non-covalentlyly with the amino acid of other interaction of molecules, but the amino acid of light cage lock and/or intramolecular photosensitization, the amino acid that comprises vitamin H or vitamin H analogue, such as glycosylation amino acid through sugar-substituted Serine, amino acid through other carbohydrate modification, the amino acid that contains ketone, the amino acid that comprises polyoxyethylene glycol or polyethers, amino acid through the heavy atom replacement, but but the amino acid of chemical cracking and/or photo-cleavage, when with the natural amino acid comparison, have the elongation side chain (include, but is not limited to polyethers or long chain hydrocarbon, include, but is not limited to greater than about 5 or greater than about 10 carbon) amino acid, the glycoprotein amino acid that contains through the carbon connection, redox active amino acids, the amino acid and the amino acid that comprises one or more toxic moieties that contain amino thioic acid sulfoacid.
Go for the present invention and can be used for that exemplary non-naturally encoded amino acid with water-soluble polymers reaction includes, but is not limited to have carbonyl, amino oxygen base, hydrazine, hydrazides, Urea,amino-, those amino acid of trinitride and alkyne reaction group.In certain embodiments, non-naturally encoded amino acid comprises the carbohydrate part.Described amino acid whose example comprises N-ethanoyl-L-glucosamine base-L-Serine, N-ethanoyl-L-galactosaminyl-L-Serine, N-ethanoyl-L-glucosamine base-L-Threonine, N-ethanoyl-L-glucosamine base-L-asparagine and O-epichitosamine base-L-Serine.Described amino acid whose example comprises that also wherein naturally occurring N-key or the O-key between the amino acid and carbohydrate is the uncommon covalent linkage of occurring in nature (including, but is not limited to alkene key, oxime key, thioether bond, amido linkage or the like) institute's metathetical example.Described amino acid whose example also comprises the carbohydrate that is not common in the naturally occurring protein, such as 2-deoxidation-glucose, 2-deoxy-galactose or the like.
The many non-naturally encoded amino acid that this paper provided can available from (for example) Sigma-Aldrich (St.Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany) or Peptech (Burlington, MA, USA).Those amino acid that can not buy be optionally according to provided herein or use the known standard method of those skilled in the art to synthesize.Relevant organic synthesis technology is for example referring to Fessendon and Fessendon Organic Chemistry, (1982, the 2 editions, Willard Grant Press, Boston Mass.); March's Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York); With Carey and Sundberg Advanced Organic Chemistry(the 3rd edition, A part and B part, 1990, PlenumPress, New York).Referring to U.S. Patent Application Publication case 2003/0082575 and 2003/0108885, it is incorporated herein by reference again.Except that the alpha-non-natural amino acid that contains novel side chain, go for alpha-non-natural amino acid of the present invention and also optionally comprise modified backbone structure, include, but is not limited to as illustrated by the structure of formula II and formula III,
Figure A20058004446400631
Wherein Z comprises OH, NH usually 2, SH, NH-R ' or S-R '; X and Y can be identical or different and comprise S or O usually, and R and R ' are optionally for identical or different and normally be selected from above and list and hydrogen about the same composition of the described R group of the alpha-non-natural amino acid of formula I.For example, alpha-non-natural amino acid of the present invention optionally comprises as by illustrated amino of formula II and formula III or the replacement on the carboxyl.Such alpha-non-natural amino acid includes, but is not limited to alpha-hydroxy acid, α-thioic acid sulfoacid, alpha-amino group carbothioic acid ester, includes, but is not limited to have corresponding to the side chain of 20 kinds of common natural amino acids or those amino acid of non-natural side chain.In addition, the replacement at the alpha-carbon place optionally includes, but is not limited to L-amino acid, D-amino acid or the α-α-dibasic amino acid such as D-L-glutamic acid, D-L-Ala, D-methyl-O-tyrosine, aminobutyric acid or the like.Other structure surrogate comprises such as the cyclic amino acid of proline analogs and 3,4,6,7,8 and 9 yuan of loop proline analogues, such as the beta amino acids and the γ amino acid of Beta-alanine that is substituted and γ-An Jidingsuan.
Many alpha-non-natural amino acids are based on the natural amino acid such as tyrosine, glutamine, phenylalanine or the like, and are applicable to the present invention.The tyrosine analogue includes, but is not limited to the tyrosine of para-orientation, the tyrosine of ortho position replacement and the tyrosine that a position replaces, and the tyrosine that wherein is substituted comprises (including, but is not limited to) ketone group (including, but is not limited to ethanoyl), benzoyl, amino, diazanyl, azanol base, thiol group, carboxyl, sec.-propyl, methyl, C 6-C 20Straight or branched alkyl, saturated or unsaturated alkyl, O-methyl, polyether group, nitro, alkynyl or the like.In addition, also contain through polysubstituted aryl rings.Go for glutamine analogue of the present invention and include, but is not limited to derivative, the cyclic derivatives of Alpha-hydroxy derivative, γ-replacement and the glutamine derivative that replaces through acid amides.The example that goes for phenylalanine analogues of the present invention includes, but is not limited to the phenylalanine of the phenylalanine of para-orientation, ortho position replacement and the phenylalanine that a position replaces, and wherein substituting group comprises (including, but is not limited to) hydroxyl, methoxyl group, methyl, allyl group, aldehyde radical, azido-, iodo, bromo, ketone group (including, but is not limited to ethanoyl), benzoyl, alkynyl or the like.The particular instance that goes for alpha-non-natural amino acid of the present invention includes, but is not limited to ethanoyl-L-phenylalanine; O-methyl-L-tyrosine; L-3-(2-naphthyl) L-Ala; the 3-methylphenylalanine; O-4-allyl group-L-tyrosine; 4-propyl group-L-tyrosine; three-O-ethanoyl-GlcNAc β-Serine; L-Dopa; fluoridize phenylalanine; sec.-propyl-L-phenylalanine; to azido--L-phenylalanine; to acyl group-L-phenylalanine; to benzoyl-L-phenylalanine; the L-phosphoserine; the phosphono Serine; phosphono tyrosine; to iodophenylalanine; to bromophenyl alanine; to amino-L-phenylalanine; sec.-propyl-L-phenylalanine and to propargyloxy-phenylalanine or the like.The example that goes for the structure of various alpha-non-natural amino acids of the present invention provides in (for example) title among the WO2002/085923 of " In vivoincorporation of unnatural amino acids. ".With regard to other methionine(Met) analogue, also can be referring to people such as Kiick, (2002) Incorporationof azides into recombinant proteins for chemoselective modification by the Staudingerligation, PNAS99:19-24, it is incorporated herein by reference.
In one embodiment, provide comprise alpha-non-natural amino acid (such as, right-(propargyloxy)-phenylalanine) GH (for example, the hGH) composition of polypeptide.Also provide and comprise right-(propargyloxy)-phenylalanine and include, but is not limited to protein and/or the various compositions of cell.On the one hand, the composition that comprises right-(propargyloxy)-phenylalanine alpha-non-natural amino acid further comprises quadrature tRNA.Alpha-non-natural amino acid can (include, but is not limited to covalently) bond mutually with quadrature tRNA; its include, but is not limited to by amino-acyl bond covalently with quadrature tRNA bond mutually, covalently with 3 ' OH of the terminal ribose of quadrature tRNA or 2 ' OH bond or the like mutually.
Provide various advantages and processing via the chemical part that can incorporate the alpha-non-natural amino acid in the protein into to protein.For example, ketone group functional group unique reactive allows to contain hydrazine or contain any reagent in the azanol reagent and in vivo to the protein sex modification that elects in vitro with many.Heavy atom alpha-non-natural amino acid (for example) can be used for phasing x-ray structure data.Use alpha-non-natural amino acid site specific ground to introduce heavy atom also provides selected location as heavy atom selectivity and handiness.Photoreactivity alpha-non-natural amino acid (including, but is not limited to have the amino acid of benzophenone and aromatic yl azide (including, but is not limited to azidomethyl phenyl nitrogenize thing) side chain) (for example) is allowed proteinic effectively in vivo and in vitro photocrosslinking.The example of photoreactivity alpha-non-natural amino acid includes, but is not limited to the triazobenzene L-Ala with to the benzoyl phenylalanine.Protein with photoreactivity alpha-non-natural amino acid therefore can be optionally crosslinked by the exciting-providing Instantaneous Control of photoreactive group.In an example, the methyl of alpha-non-natural amino acid can be through isotope-labeled (including, but is not limited to) methyl substituted, local structure and dynamic (dynamical) probe that described isotope-labeled (including, but is not limited to) methyl uses with nucleus magnetic resonance and vibrational spectroscopy as (including, but is not limited to).Alkynyl or azido-functional group (for example) allow by [3+2] cycloaddition reaction molecular selectivity ground modifying protein.
Incorporating non-natural amino acid in the polypeptide at aminoterminal can be included as and be different from 20 kinds of natural amino acids used substituent any substituent R group and be different from the NH that is present in usually in the a-amino acid (referring to formula I) 2Second reactive group of group.Similarly alpha-non-natural amino acid can be incorporated at carboxyl terminal, and it has and is different from second reactive group that is present in the COOH group in the a-amino acid (referring to formula I) usually.
Alpha-non-natural amino acid of the present invention can be selected or be designed and is provided at the further feature that is difficult to obtain in 20 kinds of natural amino acids.For example, alpha-non-natural amino acid can optionally be designed or be selected and be changed described alpha-non-natural amino acid to be incorporated into wherein proteinic biological property.For example, following character can optionally change by alpha-non-natural amino acid is contained in the protein: toxicity, bio distribution, solvability, stability are (for example, short stability to degradation of thermostability, stability to hydrolysis, oxidative stability, antienzyme or the like), purify and easiness, textural property, spectral quality, chemistry and/or the actinism handled, catalytic activity, redox potential, transformation period, ability of reacting with other molecule (for example, covalently or non-covalent ground) or the like.
The structure of alpha-non-natural amino acid (containing carbonyl, class carbonyl, the carbonyl of sheltering, shielded carbonyl and azanol base) With synthetic
In certain embodiments, the invention provides that a kind of (for example, the GH that PEG) is connected (for example, hGH) by oxime key and water-soluble polymers.
The non-naturally encoded amino acid of many types is suitable for forming the oxime key.These amino acid include, but is not limited to contain the non-naturally encoded amino acid of carbonyl, dicarbapentaborane or azanol base.Described amino acid is to be described in U.S. patent application case the 60/638th, No. 418, the 60/638th, No. 527 and the 60/639th, in No. 195 (title is " Compositions containing; methods involving; and uses of non-natural amino acids and polypeptides ", application on December 22nd, 2004), the full text of described application case is incorporated herein by reference.Described amino acid also is described in U.S. patent application case the 60/696th, No. 210, the 60/696th, No. 302 and the 60/696th, in No. 068 (title is " Compositionscontaining; methods involving; and uses of non-natural amino acids and polypeptides ", application on July 1st, 2005), the full text of described application case is incorporated herein by reference.Non-naturally encoded amino acid also is described in the U.S. patent application case the 10/126th of application on April 19th, 2002, the U.S. patent application case the 10/126th of No. 931 and on April 19th, 2002 application, in No. 927, the full text of described application case is incorporated herein by reference.
Some embodiments of the present invention are utilized the GH that through amino acid acetylphenylalanine replaced in one or more positions (for example, hGH) polypeptide.Right-ethanoyl-(+/-)-phenylalanine and-ethanoyl-(+/-)-the synthetic of phenylalanine is to be described in the Zhang that incorporates into by reference, Z. waits the people, among the Biochemistry 42:6735-6746 (2003).Other amino acid that contains carbonyl or dicarbapentaborane can be prepared similarly by the those skilled in the art.In addition, provide the non-restrictive illustrative that is included in alpha-non-natural amino acid herein synthetic in Fig. 4, Figure 24-34 of No. the 10/126th, 931, U.S. patent application case and Figure 36-39, the full text of described application case is incorporated herein by reference.
Amino acid with electrophilic reactivity group is allowed and various reactions take place and is connected with molecule, especially is connected with molecule by nucleophilic addition.Described electrophilic reactivity group comprises carbonyl (comprising ketone group and dicarbapentaborane), class carbonyl (its have similar to carbonyl (comprising ketone group and dicarbapentaborane) reactive and structurally similar to carbonyl), the carbonyl of sheltering (it can easily be converted into carbonyl (comprising ketone group and dicarbapentaborane)) or shielded carbonyl (its go have and the similar reactivity of carbonyl (comprising ketone group and dicarbapentaborane) after the protection).Described amino acid comprises the have formula amino acid of structure of (IV),
Figure A20058004446400661
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (RR ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
J is
Figure A20058004446400671
Or
Figure A20058004446400672
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
Each R " be H, alkyl, the alkyl that is substituted or protecting group independently, or " when group exists, two R " optionally forms Heterocyclylalkyl as an above R;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3And R 4In each person be H, halogen, low-carbon alkyl or the low-carbon alkyl that is substituted independently, or R 3And R 4Or two R 3Group optionally forms cycloalkyl or Heterocyclylalkyl;
Perhaps-the A-B-J-R group forms two ring or tricyclic naphthenes base or Heterocyclylalkyls of the carbonyl (comprising the dicarbapentaborane of sheltering) that comprises at least one carbonyl (comprising dicarbapentaborane), shielded carbonyl (comprising shielded dicarbapentaborane) or shelter together;
Perhaps-the J-R group forms monocycle or the bicyclic ring alkyl or the Heterocyclylalkyl of the carbonyl (comprising the dicarbapentaborane of sheltering) that comprises at least one carbonyl (comprising dicarbapentaborane), shielded carbonyl (comprising shielded dicarbapentaborane) or shelter together;
Its restricted condition is phenylene and each R for working as A 3During for H, B exists; With as A be-(CH 2) 4-and each R 3During for H, B is not-NHC (O) (CH 2CH 2)-; With as A and B does not exist and each R 3During for H, R is not a methyl.
In addition, comprise the amino acid of structure with formula V,
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Its restricted condition is for when A is phenylene, and B is what exist; With as A be-(CH 2) 4In-time, B is not-NHC (O) (CH 2CH 2)-; With when A and B do not exist, R is not a methyl.
In addition, comprise the have formula amino acid of structure of (VI),
Wherein:
B is the connexon that is selected from the group that is made up of following each group: the assorted alkylene of lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC), the assorted alkylene of low-carbon (LC) that is substituted ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Each R aBe be independently selected from the group that forms by following each group: H, halogen, alkyl, the alkyl that is substituted ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein each R ' is H, alkyl or the alkyl that is substituted independently.
In addition, comprise following amino acid:
Wherein said compound optionally is amino shielded group, the shielded compound or its salt of carboxyl.In addition, in the following alpha-non-natural amino acid any can be incorporated in the non-natural amino acid polypeptides.
In addition, comprise following amino acid with structure of formula (VII),
Figure A20058004446400692
Wherein:
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Each R aBe be independently selected from the group that forms by following each group: H, halogen, alkyl, the alkyl that is substituted ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein each R ' is H, alkyl or the alkyl that is substituted independently; And n is 0 to 8;
Its restricted condition is for as A being-(CH 2) 4In-time, B is not-NHC (O) (CH 2CH 2)-.
In addition, comprise following amino acid:
Figure A20058004446400701
With
Wherein said compound optionally is amino shielded compound, optionally is the shielded compound of carboxyl, optionally is the amino protected and shielded compound of carboxyl, or its salt.In addition, in these alpha-non-natural amino acids and the following alpha-non-natural amino acid any can be incorporated in the non-natural amino acid polypeptides.
In addition, comprise following amino acid with structure of formula (VIII),
Wherein A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.In addition, comprise following amino acid with structure of formula (IX),
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Each R wherein aBe be independently selected from the group that forms by following each group: H, halogen, alkyl, the alkyl that is substituted ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein each R ' is H, alkyl or the alkyl that is substituted independently.
In addition, comprise following amino acid:
Figure A20058004446400731
, wherein said compound optionally is amino shielded compound, optionally is the shielded compound of carboxyl, optionally is the amino protected and shielded compound of carboxyl, or its salt.In addition, in these alpha-non-natural amino acids and the following alpha-non-natural amino acid any can be incorporated in the non-natural amino acid polypeptides.
In addition, comprise following amino acid with structure of formula (X),
Figure A20058004446400732
Wherein B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Each R aBe be independently selected from the group that forms by following each group: H, halogen, alkyl, the alkyl that is substituted ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein each R ' is H, alkyl or the alkyl that is substituted independently; And n is 0 to 8.
In addition, comprise following amino acid:
Figure A20058004446400741
With
Figure A20058004446400742
Wherein said compound optionally is amino shielded compound, optionally is the shielded compound of carboxyl, optionally is the amino protected and shielded compound of carboxyl, or its salt.In addition, in these alpha-non-natural amino acids and the following alpha-non-natural amino acid any can be incorporated in the non-natural amino acid polypeptides.
Except that mono carbonyl structure, alpha-non-natural amino acid as herein described can comprise the group such as dicarbapentaborane, class dicarbapentaborane, the dicarbapentaborane of sheltering and shielded dicarbapentaborane.
For example, comprise following amino acid with structure of formula (XI),
Figure A20058004446400743
Wherein A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.
In addition, comprise following amino acid with structure of formula (XII),
Figure A20058004446400751
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Each R wherein aBe be independently selected from the group that forms by following each group: H, halogen, alkyl, the alkyl that is substituted ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein each R ' is H, alkyl or the alkyl that is substituted independently.
In addition, comprise following amino acid:
Figure A20058004446400761
With
Figure A20058004446400762
Wherein said compound optionally is amino shielded compound, optionally is the shielded compound of carboxyl, optionally is the amino protected and shielded compound of carboxyl, or its salt.In addition, in these alpha-non-natural amino acids and the following alpha-non-natural amino acid any can be incorporated in the non-natural amino acid polypeptides.
In addition, comprise following amino acid with structure of formula (XIII),
Figure A20058004446400763
Wherein B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: the low-carbon (LC) that lower alkylene, the lower alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, low-carbon (LC) mix alkylene, be substituted mix alkylene ,-O-,-O-(alkylene or the alkylene that is substituted)-,-S-,-S-(alkylene or the alkylene that is substituted)-,-S (O) k-(wherein k is 1,2 or 3),-S (O) k (alkylene or the alkylene that is substituted)-,-C (O)-,-C (O)-(alkylene or the alkylene that is substituted)-,-C (S)-,-C (S)-(alkylene or the alkylene that is substituted)-,-N (R ')-,-NR '-(alkylene or the alkylene that is substituted)-,-C (O) N (R ')-,-CON (R ')-(alkylene or the alkylene that is substituted)-,-CSN (R ')-,-CSN (R ')-(alkylene or the alkylene that is substituted)-,-N (R ') CO-(alkylene or the alkylene that is substituted)-,-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 each R ' is H, alkyl or the alkyl that is substituted independently;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Each R aBe be independently selected from the group that forms by following each group: H, halogen, alkyl, the alkyl that is substituted ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein each R ' is H, alkyl or the alkyl that is substituted independently; And n is 0 to 8.
In addition, comprise following amino acid:
With
Figure A20058004446400772
Wherein said compound optionally is amino shielded compound, optionally is the shielded compound of carboxyl, optionally is the amino protected and shielded compound of carboxyl, or its salt.In addition, in these alpha-non-natural amino acids and the following alpha-non-natural amino acid any can be incorporated in the non-natural amino acid polypeptides.
In addition, comprise following amino acid with structure of formula (XIV),
Figure A20058004446400773
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And alkylene, N (R ') (alkylene) or N (R ') (alkylene that is substituted) that L is alkylene, be substituted, wherein R ' is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted.
In addition, comprise following amino acid with structure of formula (XIV-A),
Figure A20058004446400781
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Alkylene, N (R ') (alkylene) or N (R ') (alkylene that is substituted) that L is alkylene, be substituted, wherein R ' is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted.
In addition, comprise following amino acid with structure of formula (XIV-B),
Figure A20058004446400791
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Alkylene, N (R ') (alkylene) or N (R ') (alkylene that is substituted) that L is alkylene, be substituted, wherein R ' is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted.
In addition, comprise following amino acid with structure of formula (XV),
Figure A20058004446400792
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And n is O, 1,2,3,4 or 5; And each CR 8R 9R on the group 8And R 9Each is independently selected from the group that is made up of following each group naturally: H, alkoxyl group, alkyl amine group, halogen, alkyl, aryl, or wherein any R 8And R 9Can form together=O or cycloalkyl, or wherein any group and contiguous R 8Group can form cycloalkyl together.
In addition, comprise following amino acid with structure of formula (XV-A),
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
N is 0,1,2,3,4 or 5; And each CR 8R 9R on the group 8And R 9Each is independently selected from the group that is made up of following each group naturally: H, alkoxyl group, alkyl amine group, halogen, alkyl, aryl, or wherein any R 8And R 9Can form together=O or cycloalkyl, or wherein any group and contiguous R 8Group can form cycloalkyl together.
In addition, comprise following amino acid with structure of formula (XV-B),
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
N is 0,1,2,3,4 or 5; And each CR 8R 9R on the group 8And R 9Each is independently selected from the group that is made up of following each group naturally: H, alkoxyl group, alkyl amine group, halogen, alkyl, aryl, or wherein any R 8And R 9Can form together=O or cycloalkyl, or wherein any group and contiguous R 8Group can form cycloalkyl together.
In addition, comprise following amino acid with structure of formula (XVI),
Figure A20058004446400812
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And alkylene, N (R ') (alkylene) or N (R ') (alkylene that is substituted) that L is alkylene, be substituted, wherein R ' is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted.
In addition, comprise following amino acid with structure of formula (XVI-A),
Figure A20058004446400821
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Alkylene, N (R ') (alkylene) or N (R ') (alkylene that is substituted) that L is alkylene, be substituted, wherein R ' is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted.
In addition, comprise following amino acid with structure of formula (XVI-B),
Figure A20058004446400831
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
R is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Alkylene, N (R ') (alkylene) or N (R ') (alkylene that is substituted) that L is alkylene, be substituted, wherein R ' is H, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted.
In addition, comprise the have formula amino acid of structure of (XVII),
Figure A20058004446400832
Wherein:
A is optionally, and when exist, be lower alkylene, the assorted alkylene of lower alkylene, low-carbon (LC) cyclic alkylene, the low-carbon (LC) cyclic alkylene that is substituted, low-carbon (LC) alkylene group, the low-carbon (LC) alkylene group that is substituted, alkynylene, low-carbon (LC), the assorted alkylene that is substituted that are substituted, hang down carbon heterocyclic alkylene, the low carbon heterocyclic alkylene, arylidene, the arylidene that is substituted, heteroarylidene, the heteroarylidene that is substituted, alkarylene, the alkarylene that is substituted, the inferior aralkyl that are substituted or the inferior aralkyl that is substituted;
M is
Wherein (a) expression and the bond of A group, and (b) bond of expression and corresponding carbonyl, R 3And R 4Be the cycloalkyl that is independently selected from H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or is substituted, or R 3And R 4Or two R 3Group or two R 4Group optionally forms cycloalkyl or Heterocyclylalkyl;
R is H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.
In addition, comprise the have formula amino acid of structure of (XVIII),
Figure A20058004446400842
Wherein:
M is-C (R 3)-,
Figure A20058004446400851
Wherein (a) expression and the bond of A group, and (b) bond of expression and corresponding carbonyl, R 3And R 4Be the cycloalkyl that is independently selected from H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or is substituted, or R 3And R 4Or two R 3Group or two R 4Group optionally forms cycloalkyl or Heterocyclylalkyl;
R is H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Each R aBe be independently selected from the group that forms by following each group: H, halogen, alkyl, the alkyl that is substituted ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein each R ' is H, alkyl or the alkyl that is substituted independently.
In addition, comprise the have formula amino acid of structure of (XIX),
Figure A20058004446400852
Wherein:
R is H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted; And
T 3Be O or S.
In addition, comprise the have formula amino acid of structure of (XX),
Wherein:
R is H, halogen, alkyl, the alkyl that is substituted, cycloalkyl or the cycloalkyl that is substituted.
In addition, comprise following amino acid with structure of formula (XXI):
Figure A20058004446400862
With
Figure A20058004446400863
Right-ethanoyl-(+/-)-phenylalanine and-ethanoyl-(+/-)-the synthetic of phenylalanine is to be described in the Zhang that incorporates into by reference, people such as Z. are among the Biochemistry 42:6735-6746 (2003).Other amino acid that contains carbonyl or dicarbapentaborane can be prepared similarly by the those skilled in the art.In addition, in Fig. 4, Figure 24-34 and Figure 36-39, provide the non-restrictive illustrative of the included alpha-non-natural amino acid of this paper synthetic.
In certain embodiments, the polypeptide that comprises alpha-non-natural amino acid is carried out chemically modified to produce reactive carbonyl or dicarbapentaborane functional group.For example, can be used for the aldehyde functional group of association reaction can be by the functional group's generation with contiguous amino and hydroxyl.At bioactive molecules is under the situation of polypeptide, for example, can use N terminal filament propylhomoserin or Threonine (it can normally exist maybe and can be exposed by chemistry or enzymatic digestion) to produce aldehyde functional group under the mild oxidation cracking condition that uses periodate.Referring to, for example, people such as Gaertner, Bioconjug.Chem.3:262-268 (1992); Geoghegan, K.﹠amp; Stroh, J., Bioconjug.Chem.3:138-146 (1992); People such as Gaertner, J.Biol.Chem.269:7224-7230 (1994).Yet known method is only limited to the amino acid at peptide or proteinic N end place in this technology.
In the present invention, the alpha-non-natural amino acid with contiguous hydroxyl and amino can be incorporated in the polypeptide as " sheltering " aldehyde functional group.For example, the 5-oxylysine has the hydroxyl adjacent with ε amine.The reaction conditions that is used to produce aldehyde is generally comprised within the sodium metaperiodate that adds molar excess under the mild conditions and to avoid other position in polypeptide oxidation takes place.The pH value of oxidizing reaction is generally about 7.0.The sodium metaperiodate that typical reaction comprises about 1.5 molar excess adds in the buffered soln of polypeptide, then in the dark cultivates about 10 minutes.Referring to, for example, United States Patent (USP) the 6th, 423, No. 685.
Carbonyl or dicarbapentaborane functional group can optionally react to form corresponding oxime key stable under physiological condition with the reagent that contains azanol in the aqueous solution under the condition of gentleness.Referring to, for example, 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).And unique reactivity of carbonyl or dicarbapentaborane is allowed under the situation that other amino acid side chain exists and is carried out selective modification.Referring to, for example, Cornish, people such as V. W., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F.﹠amp; Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, people such as L K., Science276:1125-1128 (1997).
The structure of alpha-non-natural amino acid (amino acid that contains azanol) and synthetic
No. the 60/638th, 418, U.S. Provisional Patent Application case be to incorporate this paper by reference in full.Thereby, in U.S. Provisional Patent Application case the 60/638th, No. 418 V chapters and sections (title is " Non-natural Amino Acids "), the disclosure that provides in the B part (title is " Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids ") is applicable to preparation fully, purifying, characterize and use alpha-non-natural amino acid as herein described, the method of non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition (comprising formula I-XXXV), technology and strategy, described how appropriate are fully by providing herein as described disclosure just.
The chemosynthesis of alpha-non-natural amino acid
Be applicable to that many alpha-non-natural amino acids of the present invention can buy, (for example) can available from Sigma (USA) or Aldrich (Milwaukee, WI, USA).Those amino acid that can not buy be optionally according to provided herein or according to provided in the various open cases or use the known standard method of those skilled in the art to synthesize.Relevant organic synthesis technology, referring to, for example, Fessendon and Fessendon's Organic Chemistry(1982, the 2 editions, Willard Grant Press, Boston Mass.); March's Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York); With Carey and Sundberg Advanced Organic Chemistry(the 3rd edition, A part and B part, 1990, Plenum Press, New York).Other open case of synthetic of describing alpha-non-natural amino acid comprises the WO2002/085923 of (for example) title for " In vivo incorporation of Unnatural Amino Acids "; People such as Matsoukas, (1995) J.Med.Chem., 38,4660-4669; King, F.E.﹠amp; Kidd, D.A.A. (1949) A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J.Chem.Soc, 3315-3319; Friedman, O.M.﹠amp; Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents. J.Am. Chem.Soc.81,3750-3752; Craig, people such as J.C. (1988) Absolute Configuration of theEnantiomers of 7-Chloro-4[[-(diethylamino)-1-methylbutyl] amino] quinoline (Chloroquine). J.Org.Chem.53,1167-1170; Azoulay, M., Vilmont, M.﹠amp; Frappier, F. (1991) Glutamineanalogues as Potential Antimalarials, Eur.J.Med.Chem.26,201-5; Koskinen, A.M.P.﹠amp; Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J.Org.Chem.54,1859-1866; Christie, B.D.﹠amp; Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the TotalSynthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium IonCyclization. J.Org.Chem.50:1239-1246; People such as Barton, (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistiy:Synthesis of L-andD-alpha-Amino-Adipfc Acids, L-alpha-aminopimelic Acid and Appropriate UnsaturatedDerivatives. Tetrahedron43:4297-4308; With people such as Subasinghe, (1992) Quisqualic acidanalogues:synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and theiractivity at a novel quisqualate-sensitized site. J.Med.Chem.35:4602-7.Also can be No. 2004/0198637, the U.S. Patent Publication case US of " Protein Arrays " referring to title, it be to be incorporated herein by reference.
A. carbonyl reaction group
Amino acid with carbonyl reaction group is allowed and various reactions take place and is connected with molecule (including but not limited to PEG or other water soluble molecules), especially is connected with described molecule by nucleophilic addition(Adn) or aldol reaction.
The exemplary amino acid that contains carbonyl can be expressed as follows,
Figure A20058004446400881
Wherein n is 0-10; R 1Be alkyl, aryl, the alkyl that is substituted or the aryl that is substituted; R 2Be H, alkyl, aryl, the alkyl that is substituted and the aryl that is substituted; And R 3Be H, amino acid, polypeptide or aminoterminal modification group, and R 4Be H, amino acid, polypeptide or carboxyl terminal modification group.In certain embodiments, n is 1, R 1Be phenyl, and R 2Be simple alkyl (that is, methyl, ethyl or propyl group), and ketone partly is in the contraposition that is positioned at respect to alkyl group side chain.In certain embodiments, n is 1, R 1Be phenyl, and R 2Be simple alkyl (that is, methyl, ethyl or propyl group), and ketone is positioned at partly with respect on the position between alkyl group side chain.
Right-ethanoyl-(+/-)-phenylalanine and/-ethanoyl-(+/-)-the synthetic of phenylalanine is to be described in people such as Zhang, Z., among the Biochemistry 42:6735-6746 (2003), it is incorporated herein by reference.Other amino acid that contains carbonyl can be prepared similarly by the those skilled in the art.
In certain embodiments, carry out chemically modified to produce reactive carbonyl functional group to comprising non-naturally encoded amino acid whose polypeptide.For example, can be used for the aldehyde functional group of association reaction can be by the functional group's generation with contiguous amino and hydroxyl.At bioactive molecules is under the situation of polypeptide, for example, can use N terminal filament propylhomoserin or Threonine (it can normally exist maybe and can be exposed by chemistry or enzymatic digestion) to produce aldehyde functional group under the mild oxidation cracking condition that uses periodate.Referring to, for example, people such as Gaertner, Bioconjug.Chem.3:262-268 (1992); Geoghegan, K.﹠amp; Stroh, J., Bioconjug.Chem.3:138-146 (1992); People such as Gaertner, J. Biol.Chem.269:7224-7230 (1994).Yet known method is only limited to the amino acid at peptide or proteinic N end place in this technology.
In the present invention, the non-naturally encoded amino acid with contiguous hydroxyl and amino can be incorporated in the polypeptide as " sheltering " aldehyde functional group.For example, the 5-oxylysine has the hydroxyl adjacent with ε amine.The reaction conditions that is used to produce aldehyde is generally comprised within the sodium metaperiodate that adds molar excess under the mild conditions and to avoid other position in polypeptide oxidation takes place.The pH value of oxidizing reaction is generally about 7.0.The sodium metaperiodate that typical reaction comprises about 1.5 molar excess adds in the buffered soln of polypeptide, then in the dark cultivates about 10 minutes.Referring to, for example, United States Patent (USP) the 6th, 423, No. 685, it is incorporated herein by reference.
The carbonyl functional group can be under mild conditions in the aqueous solution with contain hydrazine, hydrazides, azanol or Urea,amino-reagent selectivity ground react to form accordingly hydrazone key, oxime key or semicarbazone key stable under physiological condition respectively.Referring to, for example, 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).And unique reactivity of carbonyl is allowed under the situation that other amino acid side chain exists and is carried out selective modification.Referring to, for example, Cornish, people such as V.W., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F.﹠amp; Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, people such as L. K., Science 276:1125-1128 (1997).
B. hydrazine, hydrazides or Urea,amino-reactive group
Contain nucleophilic group (such as, hydrazine, hydrazides or Urea,amino-) non-naturally encoded amino acid allow and react with various electrophilic groups and form binding substances (include but not limited to and PEG or the reaction of other water-soluble polymers form binding substances).
The exemplary amino acid that contains hydrazine, hydrazides or Urea,amino-part can be expressed as follows,
Figure A20058004446400891
Wherein n is 0-10; R 1For alkyl, aryl, the alkyl that is substituted or the aryl that is substituted or do not exist; X is 0, N or S or do not exist; R 2Be H, amino acid, polypeptide or aminoterminal modification group, and R 3Be H, amino acid, polypeptide or carboxyl terminal modification group.
In certain embodiments, n is 4, R 1Do not exist, and X is N.In certain embodiments, n is 2, R 1Do not exist, and X does not exist.In certain embodiments, n is 1, R 1Be phenyl, X is O, and Sauerstoffatom is the contraposition that is positioned at the aliphatic group on the aryl rings.
The amino acid that contains hydrazides, hydrazine and Urea,amino-part can obtain from commercial source.For example, L-L-glutamic acid-γ-hydrazides can available from Sigma Chemical (St.Louis, MO).Other amino acid that can not buy can be prepared by the those skilled in the art.Referring to, for example, United States Patent (USP) the 6th, 281, No. 211, it is incorporated herein by reference.
Contain have hydrazides, the non-naturally encoded amino acid whose polypeptide of hydrazine or Urea,amino-functional group can contain aldehyde or other molecule with similar chemically reactive functional group reacts effectively and selectively with various.Referring to, for example, Shao, J. and Tam, J., J.Am.Chem.Soc.117:3893-3899 (1995).Unique reactivity of hydrazides, hydrazine and Urea,amino-functional group make its when with 20 kinds of common amino acids on the nucleophilic group (including, but is not limited to the amino of the hydroxyl of Serine or Threonine or Methionin and N end) that exists have more noticeable response for aldehyde, ketone and other electrophilic group when comparing.
C. the amino acid that contains the amino oxygen base
The non-naturally encoded amino acid that contains amino oxygen base (being also referred to as the azanol base) is allowed and is reacted with various electrophilic groups and form binding substances (include, but is not limited to and PEG or the reaction of other water-soluble polymers form binding substances).As hydrazine, hydrazides and Urea,amino-, the enhanced nucleophilicity of amino oxygen base allows it and variously contains aldehyde or other molecule with similar chemically reactive functional group reacts effectively and selectively.Referring to, for example, 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 with the reaction product of diazanyl be corresponding hydrazone, yet oxime is generally produced by the reaction of amino oxygen base with the group that contains carbonyl (such as, ketone group).
The exemplary amino acid that contains the amino oxygen base can be expressed as follows,
Wherein n is 0-10; R 1For alkyl, aryl, the alkyl that is substituted or the aryl that is substituted or do not exist; X is O, N, S or does not exist; M is 0-10; Y is C (O) or does not exist; R 2Be H, amino acid, polypeptide or aminoterminal modification group, and R 3Be H, amino acid, polypeptide or carboxyl terminal modification group.In certain embodiments, n is 1, R 1Be phenyl, X is O, and m is 1, and Y exists.Among some embodiment, n is 2, R 1Do not exist with X, m is 0, and Y does not exist.
The amino acid that contains the amino oxygen base can be prepared from the amino acid precursor (homoserine, Serine and Threonine) that is easy to obtain.Referring to, for example, M.Carrasco and R.Brown, J. Org.Chem.68:8853-8858 (2003).Some amino acid (such as, L-2-amino-4-(amino oxygen base) butyric acid) that contains the amino oxygen base is isolated (Rosenthal, G, Life Sci.60:1635-1641 (1997)) from natural origin.Other amino acid that contains the amino oxygen base can be prepared by the those skilled in the art.
D. trinitride and alkyne reaction group
Unique reactivity of trinitride and alkynes functional group makes it extremely be applicable to selective modification polypeptide and other biomolecules.Concerning general reactive electrochemical conditions, organic azide (especially being aliphatic trinitride) and alkynes are normally stable.Specifically, for 20 kinds that exist in the naturally occurring polypeptide common amino acid whose side chains (that is, the R group), trinitride and alkynes functional group are inert.Yet when entering further research, demonstrating " spring adding pressure type (the spring-loaded) " characteristic of azido-and alkynyl and its can be selectively and react by Hu Shi root [3+2] cycloaddition reaction effectively and produce corresponding triazole.Referring to, for example, people such as Chin J., Science301:964-7 (2003); Wang, people such as Q., J. Am.Chem.Soc.125,3192-3193 (2003); Chin, people such as J.W., J. Am.Chem.Soc.124:9026-9027 (2002).
Because Hu Shi root cycloaddition reaction relate to the selectivity cycloaddition reaction (referring to, for example, Padwa, A., at COMPREHENSIVE ORGANIC SYNTHESIS, the 4th volume (Trost, B.M. compile, 1991) is in the 1069-1109 page or leaf; Huisgen, R., 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (Padwa, A. compiles, and 1984), in the 1-176 page or leaf) rather than nucleophilic substitution, so have the side chain that contains trinitride and alkynyl moiety non-naturally encoded amino acid whose incorporating into allow the gained polypeptide at the non-naturally encoded selected sex modification in amino acid whose position.Relate to the GH that contains trinitride or alkynyl moiety (for example, hGH) cycloaddition reaction of polypeptide can under room temperature, the aqueous conditions by catalytic amount be used for Cu (II) in-situ reducing be Cu (I) reductive agent in the presence of add Cu (II) and (include, but is not limited to CuSO with catalytic amount 4Form) carries out.Referring to, for example, Wang, people such as Q., J. Am.Chem.Soc.125,3192-3193 (2003); Tornoe, people such as C.W., J. Org.Chem.67:3057-3064 (2002); People such as Rostovtsev, Angew.Chem.Int.Ed.41:2596-2599 (2002).Exemplary reductive agent comprises (including, but is not limited to) ascorbate salt, metallic copper, quinine (quinine), Resorcinol, vitamin K, gsh, halfcystine, Fe 2+, Co 2+With the current potential that applies.
In some cases, wherein Hu Shi root [3+2] cycloaddition reaction between trinitride and the alkynes is desired, (for example, hGH) polypeptide comprises the non-naturally encoded amino acid that comprises alkynyl moiety to GH, and will comprise the nitrine part with the water-soluble polymers that described amino acid is connected.Perhaps, also can carry out opposite reaction (that is, by part of the nitrine on the amino acid and the alkynyl moiety reaction that is present on the water-soluble polymers).
Trinitride functional group also can be optionally react with the water-soluble polymers that contains the aryl ester part and suitably by the aryl phosphine part functionalized amido linkage that produces in addition.The aryl phosphino-then reacts trinitride in-situ reducing and gained amine effectively and produces corresponding amide with the ester bond that is close to.Referring to, for example, E.Saxon and C.Bertozzi, Science 287,2007-2010 (2000).The amino acid that contains azido-can be alkyl azide (including, but is not limited to 2-amino-6-azido--1-caproic acid) or aromatic yl azide (right-azido--phenylalanine).
The exemplary water-soluble polymers that contains aryl ester and phosphine part can be expressed as follows,
Figure A20058004446400921
Wherein X can be O, N, S or does not exist, and Ph is a phenyl, and W is the aryl that water-soluble polymers and R can be H, alkyl, aryl, the alkyl that is substituted and be substituted.Exemplary R group includes, but is not limited to-CH 2,-C (CH 3) 3,-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-C (O) R ' ,-CONR ' R " ,-S (O) 2R ' ,-S (O) 2NR ' R " ,-CN and-NO 2R ', R ", R  and R " " refer to hydrogen, assorted alkyl that be substituted or that be unsubstituted, aryl that be substituted or that be unsubstituted (include, but is not limited to replace through 1-3 halogen aryl), alkyl that be substituted or that be unsubstituted, alkoxyl group or thioalkoxy group or arylalkyl independently of one another.For example, when compound of the present invention comprises a plurality of R group, as R ', R ", R  and R " " when a plurality of groups in the group exist its each selected independently naturally, each R group is selected independently.As R ' and R " when being connected with same nitrogen-atoms, its can with 5 yuan of rings of the combined formation of described nitrogen-atoms, 6 yuan the ring or 7 yuan of rings.For example ,-NR ' R " is intended to include, but is not limited to 1-pyrrolidyl and 4-morpholinyl.According to discussing above substituent, be understood by those skilled in the art that term " alkyl " is intended to comprise and comprises and the non-hydrogen group group of the carbon atom of bond mutually, (includes, but is not limited to-CF such as alkylhalide group 3With-CH 2CF 3) and acyl group (include, but is not limited to-C (O) CH 3,-C (O) CF 3,-C (O) CH 2OCH 3Or the like).
Trinitride functional group also can be optionally react with the water-soluble polymers that contains the thioesters part and suitably by the aryl phosphine part functionalized amido linkage that produces in addition.The aryl phosphino-then reacts trinitride in-situ reducing and gained amine effectively and produces corresponding amide with thioester bond.The exemplary water-soluble polymers that contains thioesters and phosphine part can be expressed as follows,
Figure A20058004446400931
Wherein n is 1-10; X can or not exist for O, N, S, and Ph is that phenyl and W are water-soluble polymers.The exemplary amino acid that contains alkynyl moiety can be expressed as follows,
Figure A20058004446400932
Wherein n is 0-10; R 1For alkyl, aryl, the alkyl that is substituted or the aryl that is substituted or do not exist; X is O, N, S or does not exist; M is 0-10, R 2Be H, amino acid, polypeptide or aminoterminal modification group, and R 3Be H, amino acid, polypeptide or carboxyl terminal modification group.In certain embodiments, n is 1, R 1Be phenyl, X does not exist, m be 0 and acetylene moiety be in the contraposition that is positioned at respect to alkyl group side chain.In certain embodiments, n is 1, R 1Be phenyl, X is O, and m is 1, and propargyloxy is (that is O-propargyl-tyrosine) in the contraposition that is positioned at respect to alkyl group side chain.In certain embodiments, n is 1, R 1Do not exist with X and m is 0 (that is PGIY).
The amino acid that contains alkynyl moiety can be buied.For example, PGIY can (Burlington MA) buys from Peptech.Perhaps, the amino acid that contains alkynyl moiety can prepare according to standard method.For example, to the propargyloxy phenylalanine can (for example) as Deiters, A. wait the people, come synthetic described in the J.Am.Chem.Soc.125:11782-11783 (2003), and 4-alkynyl-L-phenylalanine can be as Kayser, B. wait the people, Tetrahedron 53 (7): come synthetic described in the 2475-2484 (1997).Other amino acid that contains alkynyl moiety can be prepared by the those skilled in the art.
The exemplary amino acid that contains the trinitride part can be expressed as follows,
Figure A20058004446400933
Wherein n is 0-10; R 1For alkyl, aryl, the alkyl that is substituted, the aryl that is substituted or do not exist; X is O, N, S or does not exist; M is 0-10; R 2Be H, amino acid, polypeptide or aminoterminal modification group, and R 3Be H, amino acid, polypeptide or carboxyl terminal modification group.In certain embodiments, n is 1, R 1Be phenyl, X does not exist, and m is 0, and nitrine partly is to be positioned in the contraposition of alkyl group side chain.In certain embodiments, n is 0-4, and R 1Do not exist with X, and m is 0.In certain embodiments, n is 1, R 1Be phenyl, X is O, and m is 2, and β-azido-oxyethyl group partly is in the contraposition that is positioned at respect to alkyl group side chain.
The amino acid that contains the trinitride part can obtain from commercial source.For example, 4-triazobenzene L-Ala can be from Chem-Impex International, and (Wood Dale IL) obtains Inc..The amino acid that contains the trinitride part for those that can not buy; azido-can use the known standard method of those skilled in the art relatively easily to make, and includes, but is not limited to the displacement by suitable leavings group (including, but is not limited to halogenide, mesylate, tosylate group) or relatively easily makes by the open loop through the lactone of due care.Referring to, for example, March's Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York).
E. amineothiot reactive group
Make it extremely be applicable to through unique reactivity of the amineothiot functional group of beta substitution polypeptide and other biomolecules of containing aldehyde radical are carried out selective modification by forming thiazolidine.Referring to, for example, J.Shao and J.Tam, J. Am.Chem.Soc.1995,117 (14) 3893-3899.In certain embodiments, can (for example, hGH) in the polypeptide, and then react incorporate GH into through the amineothiot amino acid of beta substitution with the water-soluble polymers that comprises aldehyde functional group.In certain embodiments, water-soluble polymers, drug conjugates or other loaded article can be by forming thiazolidine and comprising (for example, hGH) the polypeptide phase coupling through the amino acid whose GH of the amineothiot of beta substitution.
The cell of alpha-non-natural amino acid absorbs
It is a problem of being considered in design with when selecting (including, but is not limited to) to be used for incorporating into the alpha-non-natural amino acid of protein usually that the alpha-non-natural amino acid of cell absorbs.For example, the high charge density of a-amino acid hints that these compounds are unlikely permeable for cell.Natural amino acid is to be absorbed in the eukaryotic cell based on proteinic haulage system by many.Can carry out rapid screening, it evaluates which kind of alpha-non-natural amino acid (if any) is absorbed by cell.Referring to, for example, at (for example) title U.S. Patent Publication case US No. 2004/0198637 (it is incorporated herein by reference) and Liu, D.R.﹠amp for " Protein Arrays "; Schultz, P.G. (1999) Progress toward theevolution of an organism with an expanded genetic code. PNAS United StatesToxicological detection among the 96:4780-4785.Though absorb and to analyze by various calibratings easily, another of alpha-non-natural amino acid that design stands cell absorption path is chosen as and provides the biosynthesizing path to produce amino acid in vivo.
(i) biosynthesizing of alpha-non-natural amino acid
Many biosynthesizing path Already in the cell to be used to produce amino acid and other compound.Though the biosynthetic means of specific alpha-non-natural amino acid may not exist nature (including, but is not limited in cell), the invention provides described method.For example, the biosynthesizing path of alpha-non-natural amino acid is to produce by adding new enzyme or changing existing host cell path in host cell.Other new enzyme optionally is the enzyme of naturally occurring enzyme or artificial development.For example, the biosynthesizing of p-Aminophenylalanine (as title for presenting in the example of the WO 2002/085923 of " In vivoincorporation of unnatural amino acids ") depends on the combination of interpolation from the known enzyme of other organism.The gene of these enzymes can be by introducing in the eukaryotic cell cell transformation with the plasmid that comprises gene.Gene provides the enzymatic path with the synthetic compound of being wanted when expressing in cell.Optionally the example of the type of the enzyme of Tian Jiaing is provided in the following example.Other enzyme sequence is found in (for example) gene pool (Genbank).Also optionally add in the cell with the enzyme of the same manner with manually development.So, the organoid of manipulated cell and resource produce alpha-non-natural amino acid.
The whole bag of tricks can be used for producing the enzyme that uses or be used to develop the novelty of existing route for the biosynthesizing path.For example, optionally will include, but is not limited to as by Maxygen, the recursiveness reorganization of Inc. exploitation (can be at www. Maxygen.comThe last acquisition) be used to develop novel enzyme and path.Referring to, for example, Stemmer (1994), Rapid evolution of aprotein 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 (can be at www. by the DesignPathTM of Genencor exploitation Genencor.comLast acquisition) is used for the metabolic pathway through engineering approaches, includes, but is not limited to be used for in order to produce the path through engineering approaches of O-methyl-L-tyrosine at cell.This technology is used the combination of new gene (those genes that include, but is not limited to differentiate by functional genome) and molecular evolution and design and rebuild existing route in host organisms.Diversa Corporation (can be at www. Diversa.comLast obtain) also be provided for the technology in rapid screening gene library and gene path, include, but is not limited to be used for producing new path.
Usually, the alpha-non-natural amino acid that produces by through engineering approaches biosynthesizing of the present invention path is to include, but is not limited to being enough to reach the biosynthesizing of effective protein proteins matter but not reaching the concentration generation of the degree that influence other amino acid whose concentration or exhaust the cell resource of n cell amount.The typical concentration that in vivo produces arrives about 0.05mM for about 10mM by this way.In case, in vivo select just optionally to be used for further optimizing the generation of the alpha-non-natural amino acid that is used for the synthetic and cell growth of ribosomal protein by after comprising plasmid in order to the gene that produces the desired enzyme of particular path and making cell transformation and produce non-natural amino acid.
(b) has the polypeptide of alpha-non-natural amino acid
Can carry out incorporating into of alpha-non-natural amino acid for the various purposes that include, but is not limited to following purpose: regulate the change of protein structure and/or function, change size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, the accessibility of proteolytic enzyme target site, part of target (including, but is not limited to the part of protein array), add bioactive molecules, connect polymkeric substance, connect radionuclide, the regulation and control serum half-life, regulation and control tissue penetration rate (for example, tumour), regulation and control active transport, regulation and control tissue, cell or organ specificity or distribution, the regulation and control immunogenicity, modulin enzyme resistance etc.The albumen mass-energy that comprises alpha-non-natural amino acid has enhanced or even brand-new catalytic property or biophysical properties.For example, following character optionally can change by alpha-non-natural amino acid is contained in the protein: toxicity, bio distribution, textural property, spectral quality, chemistry and/or actinism, catalytic capability, transformation period (including, but is not limited to serum half-life), with ability of other molecular reaction (including, but is not limited to covalently or non-covalent ground) or the like.Comprise that the proteinic composition that comprises at least one alpha-non-natural amino acid can be used for the research of (including, but is not limited to) novel therapy, diagnosis, katalaze enzyme, industrial enzyme, conjugated protein (including, but is not limited to antibody) and (including, but is not limited to) protein structure and function.Referring to, for example, Dougherty, (2000) UnnaturalAmino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645-652.
In one aspect of the invention, composition comprises at least a protein, described protein has at least 1 alpha-non-natural amino acid, includes, but is not limited to have at least 2, the alpha-non-natural amino acid more than at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 or 10.Described alpha-non-natural amino acid can be identical or different, include, but is not limited in protein, can exist comprise 1,2,3,4,5,6,7,8,1,2,3,4,5,6,7,8 of different alpha-non-natural amino acid, different position more than 9 or 10 or 10 more than 9 or 10 or 10.On the other hand, composition comprises the protein that at least one (but being less than all) in the specific amino acids that is present in the protein replaces through alpha-non-natural amino acid.Concerning specified protein with a plurality of alpha-non-natural amino acids, described alpha-non-natural amino acid can be identical or different (include, but is not limited to protein and can comprise alpha-non-natural amino acids dissimilar more than 2 or 2, maybe can comprise 2 identical alpha-non-natural amino acids).Concerning specified protein with 2 above alpha-non-natural amino acids, described alpha-non-natural amino acid can be identical, different or is the combination of the alpha-non-natural amino acid of a plurality of identical type alpha-non-natural amino acid different with at least 1.
Protein of being paid close attention to at least 1 alpha-non-natural amino acid or polypeptide are features of the present invention.The present invention also comprises having at least 1 polypeptide or protein that uses the alpha-non-natural amino acid of the compositions and methods of the invention generation.Vehicle (including, but is not limited to pharmaceutically acceptable vehicle) also can exist with protein.
Just produce protein with at least 1 alpha-non-natural amino acid or the polypeptide of being paid close attention in eukaryotic cell, described protein or polypeptide will be modified after will comprising eukaryotic translation usually.In certain embodiments, protein comprises at least 1 alpha-non-natural amino acid and at least a kind of posttranslational modification of carrying out by eukaryotic cell in vivo, and wherein said posttranslational modification is not undertaken by prokaryotic cell prokaryocyte.For example, posttranslational modification comprise that (including, but is not limited to) glycosylation, acetylizing, acylation, lipid-modified, palmitoylation effect, cetylate interpolation, phosphorylation, glycolipid key are modified, glycosylation or the like.On the one hand, posttranslational modification comprises by GlcNAc-asparagine key oligosaccharides (including, but is not limited to (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc) is connected with asparagine.Referring to table 1, some examples of the oligosaccharides that its N-that has listed eukaryotic protein connects (also can have other residue, not show in the table).On the other hand, posttranslational modification comprises by GalNAc-Serine key or GalNAc-Threonine key or GlcNAc-Serine key or GlcNAc-Threonine key oligosaccharides (including, but is not limited to Gal-GalNAc, Gal-GlcNAc etc.) is connected with Serine or Threonine.
Table 1: the example of the oligosaccharides by the GlcNAc-bonding
Figure A20058004446400971
On the other hand, posttranslational modification comprises precursor (is included, but is not limited to the thyrocalcitonin precursor, the calcitonin-gene-related peptide precursor, Pre Pro PTH (preproparathyroid) hormone, preproinsulin, proinsulin, pre-pro-opiomelanocortin (prepro-opiomelanocortin), proopiomelanocortin (pro-opiomelanocortin) etc.) carry out proteolysis processing, be assembled in the multi-subunit protein matter or carry out macromole assembling, another position of translating in the cell (includes, but is not limited to translate such as endoplasmic reticulum, golgi body (Golgi apparatus), nuclear, lysosome, peroxysome, plastosome, chloroplast(id), in the organoid of vacuole etc., or by the secretion path).In certain embodiments, protein comprises secretion sequence or positioning sequence, epitope label, FLAG label, polyhistidyl label, GST fusions or the like.The United States Patent (USP) that is incorporated herein by reference is described in detail for the 4th, 963, No. 495 and the 6th, 436, No. 674 through design and is used for improving GH (for example, hGH) the excretory construction of polypeptide.
An advantage of alpha-non-natural amino acid is that it provides other chemical part that can be used to add other molecule.These modifications can be in eukaryotic cell or non-eukaryotic cell in vivo or in vitro carrying out.Therefore, in certain embodiments, posttranslational modification is to reach by alpha-non-natural amino acid.For example, posttranslational modification can be reached by nucleophilic-electrophilic reaction.Currently be used for the proteinic most reactions of selective modification and relate between nucleophilic and electrophilic reaction collocation thing and form covalent linkage, include, but is not limited to the reaction of α-halogen ketone and Histidine or cysteine side chain.Selectivity under these situations is determined by the quantity of the nucleophilic residues in the protein and accessibility.In protein of the present invention, can adopt other to have more optionally reaction, in vitro such as and the reaction of non-natural ketone group-amino acid in vivo and hydrazides or amino oxygen based compound.Referring to, for example, people such as Cornish, (1996) J.Am.Chem.Soc, 118:8150-8151; People such as Mahal, (1997) Science, 276:1125-1128; People such as Wang, (2001) Science292:498-500; People such as Chin, (2002) J.Am.Chem.Soc.124:9026-9027; People such as Chin, (2002) Proc.Natl.Acad. Sci., 99:11020-11024; People such as Wang, (2003) Proc.Natl.Acad.Sci., 100:56-61; People such as Zhang, (2003) Biochemistry,42:6735-6746; With people such as Chin, (2003) Science,301:964-7, all documents are incorporated herein by reference.This allows with comprising that many reagent of fluorophore, linking agent, carbohydrate derivative and cytotoxic molecule come the optionally almost any protein of mark.Be No. the 6th, 927,042, the United States Patent (USP) of " Glycoprotein synthesis " referring to title also, it is incorporated herein by reference.Including, but is not limited to also can to connect (including, but is not limited to use triaryl phosphine reagent) by Staudinger (Staudinger) by the amino acid whose posttranslational modification of azido-carries out.Referring to, for example, people such as Kiick, (2002) Incorporation of azides into recombinantproteins for chemoselective modification by the Staudinger ligation, PNAS99:19-24.
The invention provides another kind selective modification method of protein efficiently, it relates in response to the alpha-non-natural amino acid of selecting codon to include, but is not limited to contain trinitride or alkynyl part and incorporating in the protein hereditarily.Then these amino acid side chains can be respectively by include, but is not limited to Hu Shi root [3+2] cycloaddition reaction (referring to, for example, Padwa, A., Comprehensive Organic Synthesis, the4 volumes, (1991), Trost, B.M. compiles, Pergamon, Oxford is in the 1069-1109 page or leaf; And Huisgen, R., 1,3-Dipolar Cycloaddition Chemistry, (1984) Padwa, A. compile, Wiley, New York is in the 1-176 page or leaf) and with including, but is not limited to alkynyl or azide derivatives is modified.Because this method relates to cycloaddition rather than nucleophilic substitution, so protein can high selectivity be modified.This reaction can be under room temperature, aqueous conditions be added in the reaction mixture by Cu (I) salt with catalytic amount and is carried out with splendid regioselectivity (1,4＞1,5).Referring to, for example, people such as Tornoe, (2002) J.Org.Chem.67:3057-3064; With people such as Rostovtsev, (2002) Angew.Chem.Int.Ed.41:2596-2599.Spendable another kind of method is the ligand exchange that utilizes on two arsenic compounds that four halfcystine motifs carry out, referring to, for example, Griffin waits the people, (1998) Science281:269-272.
Can comprise almost any molecule by the molecule that [3+2] cycloaddition joins in the protein of the present invention with trinitride or alkynyl derivatives part.Molecule includes, but is not limited to derivative, resin, bead, second protein or polypeptide (or more), polynucleotide (including, but is not limited to DNA, RNA etc.), metal chelator, cofactor, lipid acid, carbohydrate of dyestuff, fluorophore, linking agent, carbohydrate derivative, polymkeric substance (including, but is not limited to the derivative of polyoxyethylene glycol), photocrosslinking agent, cytotoxin compounds, affinity labelling, vitamin H or the like.These molecular energies join respectively in alpha-non-natural amino acid (including, but is not limited to the propargyloxy phenylalanine) with alkynyl or the alpha-non-natural amino acid (including, but is not limited to the triazobenzene L-Ala) with azido-.
V. comprise amino acid whose GH (for example, hGH) the in vivo generation of polypeptide of non-genetic coding
GH of the present invention (for example, hGH) the polypeptide amino acid that can use modified tRNA and tRNA synthetic enzyme to be added in vivo not to be encoded in the naturally occurring system or replace and produce with it.
Being used for producing the amino acid whose tRNA that use is not encoded in naturally occurring system and the method for tRNA synthetic enzyme is to be described in (for example) U.S. Patent Application Publication case 2003/0082575 the (the 10/126th, No. 927) and 2003/0108885 the (the 10/126th, No. 931) in, described reference is incorporated herein by reference.These methods relate to producing and are independent of concerning translation system the machine translator that works for endogenic (and so be known as sometimes " orthogonal ") synthetic enzyme and tRNA.Usually, translation system comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS).Usually, the amino acid that O-RS preferentially exists with at least a non-natural in translation system makes the amino acidylate of O-tRNA, and O-tRNA identifies at least a selection codon that can not be identified by other tRNA in the described system.In the protein that described translation system therefore produces non-naturally encoded aminoacid insertion in the system in response to encoded selection codon, thereby with in the position of amino acid " replacement " in the encoded polypeptide.
Various quadrature tRNA and aminoacyl tRNA synthetase have been described the synthesizing amino acid that is used for specific and have been inserted into polypeptide in affiliated field, and generally are applicable to the present invention.For example, ketone group specificity O-tRNA/ aminoacyl-tRNA synthetase is described in Wang, people such as L., and Proc.Natl.Acad.Sci.USA 100:56-61 (2003) and Zhang, people such as Z. are among Biochem.42 (22): the 6735-6746 (2003).Exemplary O-RS or its part are to encode by the polymerized nucleoside acid sequence, and it comprises the aminoacid sequence that is disclosed in U.S. Patent Application Publication case 2003/0082575 and 2003/0108885, and each open case is incorporated herein by reference.Also be described in U.S. Patent Application Publication case 2003/0082575 (the 10/126th, No. 927) and 2003/0108885 (the 10/126th, No. 931) with the corresponding O-tRNA molecule of a use of O-RS, described open case is incorporated herein by reference.
The example of azido-specificity O-tRNA/ aminoacyl-tRNA synthetase system is to be described in people such as Chin, J.W., among the J. Am.Chem.Soc.124:9026-9027 (2002).The exemplary O-RS sequence of right-azido--L-phenylalanine includes, but is not limited to as U.S. Patent Application Publication case 2003/0108885 the (the 10/126th, No. 931) middle nucleotide sequence SEQ ID NO:14-16 and 29-32 and aminoacid sequence SEQ ID NO:46-48 and the 61-64 that discloses, described open case is incorporated herein by reference.Be applicable to that exemplary O-tRNA sequence of the present invention includes, but is not limited to as U.S. Patent Application Publication case 2003/0108885 the (the 10/126th, No. 931) the middle nucleotide sequence SEQID NO:1-3 that discloses, described open case is incorporated herein by reference.To other example that it is right that specific non-naturally encoded amino acid has specific O-tRNA/ aminoacyl-tRNA synthetase is to be described in U.S. Patent Application Publication case 2003/0082575 the (the 10/126th; No. 927) in, described open case is incorporated herein by reference.Make the amino acid of ketone group containing and contain O-RS and the O-tRNA that the amino acid of azido-incorporates in the yeast saccharomyces cerevisiae (S.cerevisiae) to be described in people such as Chin, J.W., among the Science 301:964-967 (2003).
Reported that some other quadrature is right.Described the glutaminyl that is derived from yeast saccharomyces cerevisiae tRNA and synthetic enzyme (referring to, for example, Liu, D.R. and Schultz, P.G. (1999) Proc.Natl.Acad.Sci.U.S.A.96:4780-4785), aspartyl (referring to, for example, Pastrnak, people such as M., (2000) Helv.Chim.Acta83:2277-2286) and tyrosyl (referring to, for example, Ohno, people such as S., (1998) J.Biochem. (Tokyo, Jpn.)1 124:1065-1068; And Kowal, people such as A.K., (2001) Proc.Natl.Acad.Sci.U.S.A.98:2268-2273) system is used for incorporating alpha-non-natural amino acid into intestinal bacteria potentially.Described be derived from the intestinal bacteria glutaminyl (referring to, for example, Kowal, people such as A.K., (2001) Proc.Natl.Acad.Sci.U.S.A.98:2268-2273) and tyrosyl (referring to, for example, Edwards, H. and Schimmel, P. (1990) Mol.Cell. Biol.10:1633-1641) system of synthetic enzyme is applicable to yeast saccharomyces cerevisiae.Intestinal bacteria tyrosyl system is used in vivo incorporating 3-iodo-L-tyrosine into mammalian cell.Referring to, Sakamoto, people such as K., (2002) Nucleic Acids Res.30:4692-4699.
The use of O-tRNA/ aminoacyl-tRNA synthetase relates to the selection of the non-naturally encoded amino acid whose specificity codon of coding.Though can use any codon, usually need to select seldom or never to be used for to express the codon of the cell of O-tRNA/ aminoacyl tRNA synthetase.For example, exemplary codon comprises nonsense codon, such as, terminator codon (amber codon, ocher codon and opal codon); 4 or 4 above base codons; Other natural 3 base codons that seldom use or do not used.
Can use known mutation method in this technology (including, but is not limited to site specific sudden change, cassette mutagenesis, restricted selective mutation etc.) to select codon to introduce GH (for example, hGH) in the appropriate location in the polynucleotide encoding sequence specificity.
Be used to produce can be used to incorporate into non-naturally encoded amino acid whose protein biosynthesizing machine component (such as O-RS, O-tRNA and quadrature O-tRNA/O-RS to) method be to be described in people such as Wang, L., Science 292:498-500 (2001); Chin, people such as J.W., J. Am.Chem.Soc.124:9026-9027 (2002); Zhang, people such as Z. are among the Biochemistry 42:6735-6746 (2003).Being used in vivo incorporating into non-naturally encoded amino acid whose method and composition is to be described in the U.S. Patent Application Publication case 2003/0082575 (the 10/126th, No. 927), and described open case is incorporated herein by reference.The right method of quadrature tRNA-tRNA synthetic enzyme that is used to select be applicable to the in vivo translation system of organism also is described in U.S. Patent Application Publication case 2003/0082575 the (the 10/126th, No. 927) and 2003/0108885 the (the 10/126th, No. 931) in, described open case is incorporated herein by reference.Title be used to incorporate into the amino acid whose quadrature RS of ketone group for the open case WO of the PCT of " SiteSpecific Incorporation of Keto Amino Acids into Proteins " No. 04/035743 (it is incorporated herein by reference in full) describes and tRNA right.Title is used for non-naturally encoded amino acid is incorporated into eukaryotic host cell for the open case WO of the PCT of " Expanding the Eukaryotic Genetic Code " No. 04/094593 (it is incorporated herein by reference in full) describes quadrature RS and tRNA are right.
The method that is used to produce the quadrature aminoacyl-tRNA synthetase (O-RS) of at least a reorganization comprises: (a) produce the storehouse that stems from from (optionally being mutant) RS of at least a aminoacyl-tRNA synthetase (RS) of first organism, described first organism includes, but is not limited to such as Methanococcus jannaschii, horizontal river virus, Halobacterium, bacillus coli, the ancient green-ball bacterium (A.fulgidus) of glimmering, fierce fireball bacterium (P.furiosus), hole Yue Shi fireball bacterium (P horikoshii), the quick hot bacterium of gas (A.pernix) of thermophile bacteria, the prokaryotic organism of thermus thermophilus (T. thermophilus) or the like, or eukaryote; (b) select the member in (and/or screening) RS (optionally being mutant RS) storehouse, described member makes quadrature tRNA (O-tRNA) aminoacylation in the presence of non-naturally encoded amino acid and natural amino acid, thereby the set of active (optionally being mutant) RS is provided; And/or the active RS (including, but is not limited to the RS that suddenlys change) that (c) selects (optionally selecting) to gather by negativity, described active RS preferentially makes the O-tRNA aminoacylation not existing under the non-naturally encoded amino acid whose situation, thereby at least a reorganization O-RS is provided; Wherein said at least a reorganization O-RS preferentially makes the O-tRNA aminoacylation with non-naturally encoded amino acid.
In one embodiment, RS is nonactive RS.Nonactive RS can produce by making active RS sudden change.For example, nonactive RS can by make 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 produce at least about the different aminoacids that the above amino acid mutation of 10 or 10 becomes to include, but is not limited to L-Ala.
The storehouse of sudden change RS can use known various technology in this technology (include, but is not limited to the appropriate design based on the three-dimensional RS structure of protein, or in the sudden change of RS Nucleotide at random or in the appropriate design technology) to produce.For example, sudden change RS can produce recombination mutation, chimeric construct, appropriate design and produces by known other method in described or this technology herein by site specific sudden change, random mutation, difference.
In one embodiment, select (and/or screening) RS (optionally being sudden change RS) storehouse include, but is not limited to the active member of quadrature tRNA (O-tRNA) aminoacylation is comprised: will include, but is not limited to the positivity selection of antibiotics resistance gene etc. or selection markers and (optionally for sudden change) RS storehouse is introduced in a plurality of cells, wherein said positivity is selected and/or selection markers comprises the selection codon that at least one includes, but is not limited to amber codon, ocher codon or opal codon; Described a plurality of cell is grown in the presence of selective agent; Discriminating select and/or selective agent in the presence of by suppress that positivity is selected or selection markers at least one select codon survival cell of (or showing specific reaction), thereby the subclass of the cell that the positivity of the set that contains activity (optionally for sudden change) RS selectes is provided.Optionally can change and select and/or selective agent concentration.
On the one hand, the positivity selective marker is chloramphenicol acetyltransferase (CAT) gene, and the selection codon is the amber terminator codon in the CAT gene.Optionally, the positivity selective marker is the β-Nei Xiananmei gene, and the selection codon is the amber terminator codon in the β-Nei Xiananmei gene.On the other hand, the positivity selection markers comprises fluorescence or luminous selection markers or based on the selection markers (including, but is not limited to cell surface marker) of avidity.
In one embodiment, the active RS that preferentially makes the O-tRNA aminoacylation under not having non-naturally encoded amino acid whose situation (optionally being what suddenly change) that set was selected or screened to negativity comprising: negativity selection or selection markers are introduced in a plurality of cells of second organism with the set of the activity of selecting from positivity or screen (optionally for suddenling change) RS, wherein said negativity is selected or selection markers comprises at least one selection codon (include, but is not limited to antibiotics resistance gene, include, but is not limited to chloramphenicol acetyltransferase (CAT) gene); With differentiate in survival in first substratum that non-naturally encoded amino acid and screening or selective agent replenish or show the specificity screening reaction but in second substratum that replenishes without non-naturally encoded amino acid and selection or selective agent, fail to survive or do not show the cell of specific reaction, thereby provide survivaling cell or through the cell of screening with at least a reorganization O-RS.For example, the CAT authentication schemes plays that positivity is selected and/or the negativity screening in the determining of suitable O-RS recombinant chou.For example, duplicate clone's set on have or do not have one or more non-naturally encoded amino acid whose CAT if necessary in the containing grown cultures dish of (its comprise at least one select codon).Therefore think, contain reorganization O-RS containing the group of growing on the non-naturally encoded amino acid whose culture plate only.On the one hand, it is transformable selecting the concentration of (and/or screening) agent.In certain aspects, first organism is different with second organism.Therefore, first organism and/or second organism optionally comprise: prokaryotic organism, eukaryote, Mammals, bacillus coli, fungi, yeast, ancient bacterium, eubacterium, plant, insect, protobiont etc.In other embodiments, selection markers comprises fluorescence or luminous selection markers or based on the selection markers of avidity.
In another embodiment, screen or select activity (optionally being what the suddenly change) RS of (including, but is not limited to negativity selects) set to comprise: to make from positivity and select the set of the activity sudden change RS of step (b) to separate; The set of negativity selection or selection markers and activity (optionally being what suddenly change) RS is introduced in a plurality of cells of second organism, wherein said negativity is selected or selection markers comprises at least one selection codon (include, but is not limited to comprise the toxicity marker gene that at least one selects codon, include, but is not limited to rnase barnase (barnase) gene); With differentiate in without first substratum of non-naturally encoded amino acid supplementation survival or show the specificity screening reaction but in second substratum of non-naturally encoded amino acid supplementation, failing to survive or do not showing the cell of specificity screening reaction, thereby survivaling cell or the cell through screening with at least a reorganization O-RS are provided, and wherein at least a reorganization O-RS is specific for non-naturally encoded amino acid.On the one hand, at least one selects codon to comprise about selection codon more than 2 or 2.Described embodiment can comprise optionally that wherein at least one selects codon to comprise selection codon and first organism different with second organism (including, but is not limited to each organism optionally is (including, but is not limited to) prokaryotic organism, eukaryote, Mammals, bacillus coli, fungi, yeast, ancient bacterium, eubacterium, plant, insect, protobiont etc.) wherein more than 2 or 2.In addition, some aspects comprise that wherein the negativity selective marker comprises rnase barnase gene (it comprises at least one and selects codon).Others comprise that selection markers wherein optionally comprises fluorescence or luminous selection markers or based on the selection markers of avidity.Among the embodiment in this article, screening and/or the variation of selecting optionally to comprise screening and/or selecting severity.
In one embodiment, the method that produces the quadrature aminoacyl-tRNA synthetase (O-RS) of at least a reorganization can further comprise: at least a reorganization O-RS is separated; (e) produce second group of O-RS (optionally being what suddenly change) that is derived from described at least a reorganization O-RS; (f) repeating step (b) and (c) till obtaining to comprise the sudden change O-RS of the ability that preferentially makes the O-tRNA aminoacylation.Optionally repeating step (d)-(f) includes, but is not limited to repetition at least about twice.On the one hand, second group of sudden change O-RS that is derived from least a reorganization O-RS can produce by sudden change, reorganization or its combination that includes, but is not limited to random mutation, site specific sudden change.
In aforesaid method, include, but is not limited to positivity selection/screening step (b), negativity selection/screening step (c) or positivity and negativity selection/screening step (b) and (c) severity of both selection/screening steps optionally comprise and change selection/screening severity.In another embodiment, positivity selection/screening step (b), negativity selection/screening step (c) or positivity and negativity selection/screening step (b) and (c) both comprise that to use report body, wherein said report body be to detect or wherein said report body is to detect by luminous by fluorescent activation cell sorting method (FACS).Optionally, the report body is on the cell surface, show on phage display system etc., and is based on the avidity that relates to non-naturally encoded amino acid or analogue or catalytic activity and selects.In one embodiment, the synthetic enzyme of sudden change is on the cell surface, show on phage display system etc.
The method that produces the quadrature tRNA (O-tRNA) of reorganization comprises: (a) produce and stem from the storehouse of sudden change tRNA that includes, but is not limited to suppress the tRNA of tRNA from first organism at least a; (b) select (including, but is not limited to negativity selects) or screen described storehouse under not existing from the situation of the aminoacyl-tRNA synthetase (RS) of first organism by (optionally for sudden change) tRNA from the RS aminoacylation of second organism, thereby tRNA is provided the set of (optionally being mutant); (c) select or screening tRNA (optionally being mutant) set by the member of quadrature that introduced RS (O-RS) aminoacylation, thereby at least a reorganization O-tRNA is provided; Wherein said at least a reorganization O-tRNA identifies and selects codon and effectively do not identified by the RS from second organism, and by O-RS aminoacylation preferentially.In certain embodiments, at least a tRNA suppresses tRNA and/or comprises the single 3 base codons of natural and/or non-natural base, or nonsense codon, rare codon, non-natural codon, the codon that comprises at least 4 bases, amber codon, ocher codon or opal terminator codon.In one embodiment, reorganization O-tRNA has the orthogonality of improvement.Should be appreciated that in certain embodiments, O-tRNA optionally can need not to modify and promptly be input to first organism from second organism.In various embodiments, first organism is identical or different with second organism, and optionally is to be selected from (including, but is not limited to) prokaryotic organism (including, but is not limited to Methanococcus jannaschii, horizontal river virus, bacillus coli, Halobacterium etc.), eukaryote, Mammals, fungi, yeast, ancient bacterium, eubacterium, plant, insect, protobiont etc.In addition, reorganization tRNA is optionally by non-naturally encoded amino acid aminoacylation, and wherein said non-naturally encoded amino acid is in vivo to handle and in addition biosynthesizing natively or by heredity.Optionally with non-naturally encoded aminoacid addition in the growth medium of at least the first organism or second organism.
On the one hand, select (optionally for the sudden change) tRNA (step (b)) by the aminoacyl-tRNA synthetase aminoacylation in (including, but is not limited to negativity selects) or screening storehouse to comprise: toxicity marker gene and (optionally for sudden change) tRNA storehouse are incorporated in a plurality of cells from second organism, wherein said toxicity marker gene comprises at least one and selects codon (or cause the toxigenicity agent or staticize the gene of agent or be essential gene to organism, wherein said marker gene comprises at least one and selects codon); With the cell of selecting survival, the cell of wherein said survival contains the set of (optionally being what the suddenly change) tRNA that comprises at least a quadrature tRNA or non-functional tRNA.For example, the cell of survival can be selected by using the calibrating of cell density comparing rate.
On the other hand, the toxicity marker gene can comprise the selection codon more than 2 or 2.In another embodiment of described method, the toxicity marker gene is a rnase barnase gene, and wherein said rnase barnase gene comprises at least one amber codon.Optionally, rnase barnase gene can comprise the amber codon more than 2 or 2.
In one embodiment, select or the member by quadrature that introduced RS (O-RS) aminoacylation of the set of screening (optionally for sudden change) tRNA can comprise: positivity is selected or the selection markers gene is incorporated in a plurality of cells from second organism together with the set of O-RS and (optionally for sudden change) tRNA, wherein the positivity marker gene comprises drug resistance gene and (includes, but is not limited to the β-Nei Xiananmei gene, it comprises at least one and selects codon, such as at least one amber terminator codon) or organism is essential gene or causes toxic agents toxicide gene; With differentiate include, but is not limited to antibiotic selection or selective agent in the presence of the survivaling cell of growing or through the cell of screening; thereby provide the set of cell with at least a reorganization tRNA; wherein select codon in response at least one, at least a reorganization tRNA by the O-RS aminoacylation and with aminoacid insertion to being in the coded translation product of positivity marker gene.In another embodiment, the concentration of selection and/or selective agent is transformable.
Be provided for producing the right method of specificity O-tRNA/O-RS.Method comprises: (a) produce the storehouse stem from from the sudden change tRNA of at least a tRNA of first organism; (b) negativity select or screen described storehouse under not existing from the situation of the aminoacyl-tRNA synthetase (RS) of first organism by (optionally for sudden change) tRNA from the RS aminoacylation of second organism, thereby the set of (optionally for sudden change) tRNA is provided; (c) select or the set of screening (optionally for sudden change) tRNA by the member of quadrature that introduced RS (O-RS) aminoacylation, thereby at least a reorganization O-tRNA is provided.Described at least a reorganization O-tRNA identifies the selection codon and is not effectively identified by the RS from second organism, and preferentially by the O-RS aminoacylation.Described method comprises that also (d) produces the storehouse stem from from (optionally for sudden change) RS of at least a aminoacyl-tRNA synthetase (RS) of the 3rd organism; (e) select or the member who in the presence of non-naturally encoded amino acid and natural amino acid, preferentially makes at least a reorganization O-tRNA aminoacylation in screening sudden change RS storehouse, thereby the set of active (optionally for sudden change) RS is provided; (f) negativity select or screen described set do not have the activity that preferentially makes at least a reorganization O-tRNA aminoacylation under the non-naturally encoded amino acid whose situation (optionally for sudden change) RS; thereby provide at least a specificity O-tRNA/O-RS right, wherein at least a specificity O-tRNA/O-RS is specific reorganization O-RS and at least a reorganization O-tRNA to comprising at least a to non-naturally encoded amino acid.Comprise that the specificity O-tRNA/O-RS that is produced by described method is right.For example, specificity O-tRNA/O-RS is right to comprising (including, but is not limited to) mutRNATyr-mutTyrRS, such as, mutRNATyr-SS12TyrRS to, mutRNALeu-mutLeuRS to, mutRNAThr-mutThrRS to, mutRNAGlu-mutGluRS to or the like.In addition, described method comprises that wherein first organism is identical (including, but is not limited to Methanococcus jannaschii) with the 3rd organism.
The right method of quadrature tRNA-tRNA synthetic enzyme that is used for selecting being applicable to the in vivo translation system of second organism is also included within the present invention.Described method comprises: marker gene, tRNA and the aminoacyl-tRNA synthetase (RS) that separates from first organism or obtain are incorporated into first group of cell from second organism; Marker gene and tRNA are incorporated in the replicating cell group from second organism; With the cell that is chosen in survival in first group but in the replicating cell group, fails to survive, or screening shows the specificity screening reaction but do not produce the cell of described reaction in the replicating cell group, wherein first group and replicating cell group be select or selective agent in the presence of grow survivaling cell or to comprise the quadrature tRNA-tRNA synthetic enzyme of the in vivo translation system that is applicable to second organism through the cell of screening right wherein.In one embodiment, relatively and select or screening comprises in vivo complementary calibrating.Can change the concentration of selection or selective agent.
Organism of the present invention comprises various organisms and various combination.For example, first organism of method of the present invention and second organism can be identical or different.In one embodiment, organism is prokaryotic organism optionally, includes, but is not limited to Methanococcus jannaschii, horizontal river virus, Halobacterium, bacillus coli, the ancient green-ball bacterium (A.fulgidus) of flicker, fierce fireball bacterium (P.furiosus), hole Yue Shi fireball bacterium (P.horikoshii), the quick hot bacterium of gas (A.pernix) of thermophile bacteria, thermus thermophilus or the like.Perhaps, organism optionally comprises eukaryote, includes, but is not limited to plant (including, but is not limited to the complicated plant such as monocotyledons or dicotyledons), algae, protobiont, fungi (including, but is not limited to yeast etc.), animal (including, but is not limited to Mammals, insect, arthropods etc.) or the like.In another embodiment, second organism is prokaryotic organism, includes, but is not limited to Methanococcus jannaschii, horizontal river virus, Halobacterium, bacillus coli, the ancient green-ball bacterium (A.fulgidus) of flicker, Halobacterium, fierce fireball bacterium (P.furiosus), hole Yue Shi fireball bacterium (P. horikoshii), the quick hot bacterium of gas (A.pernix) of thermophile bacteria, thermus thermophilus or the like.Perhaps, second organism can be eukaryote, includes, but is not limited to yeast, zooblast, vegetable cell, fungi, mammalian cell or the like.In various embodiments, first organism is different with second organism.
VI. the amino acid that exists of non-natural is in GH (for example, the hGH) location in the polypeptide
The present invention is contained the amino acid that one or more non-naturals are existed and is incorporated GH into (for example, hGH) in the polypeptide.The amino acid that one or more non-naturals exist can be incorporated in the specific location of not destroying polypeptide active.This can replace and to realize by carrying out " conservative property ", described " conservative property " replace include, but is not limited to hydrophobic amino acid replace that hydrophobic amino acid, huge huge amino acid, the hydrophilic amino acid of aminoacid replacement replace hydrophilic amino acid and/or aminoacid insertion that non-natural is existed in the active desired position.
(for example, each district hGH) can followingly illustrate that wherein the amino acid position among the hGH is shown in (SEQ ID NO:2) in the middle row to GH.
Spiral A spiral B spiral C spiral D
[1-5]-[6-33]-[34-74]-[75-96]-[97-105]-[106-129]-[130-153]-[154-183]-[184-191]
N end A-B B-C ring C-D ring C end
Can adopt various biological chemistries and structural approach to select to be used at GH (for example, the position of being wanted that hGH) replaces with non-naturally encoded amino acid in the polypeptide.The those skilled in the art is easy to understand, any position of described polypeptide chain be suitable for selecting to incorporate non-naturally encoded amino acid into, and for any specific institute's syllabus or be not any specific institute's syllabus, selection can be carried out based on appropriate design or by selecting at random.Selecting the position of wanting can be used for generation has and anyly (for example wants character or active GH, hGH) molecule (including, but is not limited to agonist, super agonist, reverse agonist, antagonist, receptors bind conditioning agent, receptor activity modulators), being used for dimer or polymer forms, comparing with natural molecule does not change activity or character, or regulates any physical properties or the chemical property (such as solvability, aggregation or stability) of polypeptide.For example, can use scanning of known point mutation analysis in this technology, L-Ala or homologue scanning method to differentiate GH (for example, hGH) the required polypeptide position of the biological activity of polypeptide.Referring to, for example, Cunningham, B. and Wells, J., Science, 244:1081-1085 (1989) (to GH (for example identifies 14, hGH) the very crucial residue of biological activity) and Cunningham, people such as B., Science 243:1330-1336 (1989) (using homologue scanning sudden change method to differentiate antibody and receptor antigen decision base).United States Patent (USP) the 5th, 580, No. 723, the 5th, 834, No. 250, the 6th, 013, No. 478, the 6th, 428, No. 954 and the 6th, 451, describe for No. 561 by the active structure domain that utilizes the target material to differentiate to influence polypeptide active to polypeptide (such as, hGH) 26S Proteasome Structure and Function carries out the method for systems analysis, and described patent is to be incorporated herein by reference.Except that differentiating by L-Ala or homologue scanning sudden change method to can being the good candidate residue that replaces with non-naturally encoded amino acid to the residue those very crucial residues of biological activity, this on the polypeptide looked for want activity to decide.Perhaps, differentiate to also can being the good candidate position that replaces with non-naturally encoded amino acid the very crucial position of biological activity, this also on the polypeptide looked for want active and decide.Another kind of alternative will be for replacing with non-naturally encoded amino acid each position on polypeptide chain and observing influence to polypeptide active simply continuously.The those skilled in the art is easy to understand, and any way, technology or the method that are used for selecting alpha-non-natural amino acid to be substituted onto the position of any polypeptide are applicable to the present invention.
Also can study the structure and the activity of the naturally occurring mutant of the hGH polypeptide that contains disappearance and may allow the protein zone that replaces with non-naturally encoded amino acid with definite.About hGH, referring to, for example, people such as Kostyo, Biochem.Biophys.Acta, 925:314 (1987); Lewis, people such as U., J. Biol.Chem., 253:2679-2687 (1978).In a similar manner, can use protease digestion and monoclonal antibody to differentiate to undertake hGH zone in conjunction with the hGH acceptor.Referring to, for example, Cunningham, people such as B., Science 243:1330-1336 (1989); Mills, people such as J., Endocrinology, 107:391-399 (1980); Li, C., Mol.Cell.Biochem., 46:31-41 (1982) (showing that amino acid between residue 134 and 149 can lack and activity can not lost).In case after may not allowing that the residue that replaces with non-naturally encoded amino acid has been removed, just can study the influence of the replacement of on each rest position, being advised according to the three-dimensional crystalline structure of hGH and its conjugated protein.About hGH, referring to de Vos, people such as A., Science, 255:306-312 (1992); The all crystals structure of hGH can (PDB can be at www. at Protein Data Bank (Protein Data Bank) (comprising 3HHR, 1AXI and 1HWG) Rcsb.orgLast acquisition) obtain in, described Protein Data Bank is the centralized data base that contains the three-dimensional structure data of protein and nucleic acid molecule.If three-dimensional structure data can not obtain, can set up the secondary of research polypeptide and the model of tertiary structure so.Therefore, the those skilled in the art can be easy to differentiate can be through the amino acid position of non-naturally encoded aminoacid replacement.
In certain embodiments, (for example, hGH) polypeptide comprises the amino acid that the non-natural in one or more protein zones that are arranged in the spiral that can not destroy polypeptide or βZhe Die secondary structure exists to GH of the present invention.
Incorporating non-naturally encoded amino acid whose exemplary residue into can be that those get rid of the residue outside possible receptors bind zone (including, but is not limited to position I and position II), can be those residues that completely or partially are exposed to solvent, those hydrogen bondings with contiguous residue interact minimum or do not have the interactional residue of hydrogen bonding with contiguous residue, can be the residue that those minimallies are exposed to contiguous reactive residue, and can be those as by with the three-dimensional crystalline structure of its receptors bind or unconjugated hGH, secondary, residue on highly flexible (including, but is not limited to the C-D ring) that three grades or quaternary structure are predicted or the structure in inflexible (the including, but is not limited to the B spiral) zone.
In certain embodiments, incorporate into corresponding to any position amino acid that one or more are non-naturally encoded in one or more following column regions of hGH secondary structure following: corresponding to 1-5 (N end) from SEQ ID NO:2,6-33 (spiral A), 34-74 (the zone between spiral A and the spiral B, the A-B ring), 75-96 (spiral B), 97-105 (the zone between spiral B and the spiral C, the B-C ring), 106-129 (spiral C), 130-153 (zone between spiral C and the spiral D, C-D ring), 154-183 (spiral D), the position of 184-191 (C end).In other embodiments, GH polypeptide of the present invention (for example, the hGH polypeptide) comprises the amino acid that at least one non-natural exists, the aminoacid replacement that described non-natural exists (for example is positioned at GH, hGH) at least one amino acid at least one zone, described zone are to be selected from by corresponding to the group that forms with the zone of lower area: N end (32-46), B-C ring (97-105), C-D ring (132-149) and the C of the N end (1-5) of SEQ ID NO:2, A-B ring hold (184-191).In certain embodiments, one or more non-naturally encoded amino acid at GH (for example are, hGH) incorporate into one or more following position, described position corresponding to: position 1 before (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).
Incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises corresponding to the position with upper/lower positions: position 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).
The subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises corresponding to the position with upper/lower positions: position 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187 or its any combination (from SEQID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).GH (for example, hGH) crystalline structure and itself and GH are (for example, hGH) Study of Interaction of acceptor shows that the side chain of these amino-acid residues is that completely or partially to can be solvent institute approaching, and non-naturally encoded amino acid whose side chain can point in the solvent away from protein surface.
Incorporating one or more non-naturally encoded amino acid whose example location into comprises corresponding to the position with upper/lower positions: position 35,88,91,92,94,95,99,101,103,111,131,133,134,135,16,139,140,143,145 and 155 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).(for example, (for example, hGH) Study of Interaction of acceptor shows that the side chain of these amino-acid residues is fully to be exposed in side chain sensing solvent solvent and natural residue to GH with GH for crystalline structure hGH) and its.
The subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises corresponding to the position with upper/lower positions: position 30,74,103 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises corresponding to the position with upper/lower positions: position 35,92,143,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises corresponding to the position with upper/lower positions: position 35,92,131,134,143,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises corresponding to the position with upper/lower positions: position 30,35,74,92,103,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ IDNO:1 or 3).Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises corresponding to the position with upper/lower positions: position 35,92,143,145 or its any combination (from SEQID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, incorporate one or more non-naturally encoded amino acid whose positions into and comprise position corresponding to position 35 (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).
In certain embodiments, (for example, hGH) the non-naturally encoded amino acid of at least one contains carbonyl (for example, ketone group) to incorporate GH into.In certain embodiments, (for example, at least one the non-naturally encoded amino acid in hGH) is to acetylphenylalanine to incorporate GH into.(for example, hGH) contain among a plurality of non-naturally encoded more amino acid whose embodiment, (for example, a plurality of non-naturally encoded amino acid in hGH) is to acetylphenylalanine to incorporate GH at GH.(for example, hGH) contain among a plurality of non-naturally encoded more amino acid whose embodiment, (for example, all substantially non-naturally encoded amino acid in hGH) is to acetylphenylalanine to incorporate GH at GH.
In certain embodiments, the amino acid that non-natural exists is to be connected with water-soluble polymers in one or more positions that include, but is not limited to corresponding to the position of upper/lower positions: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the amino acid that non-natural exists is connected at the certain position place with water-soluble polymers, described position includes, but is not limited to the position corresponding to one or more positions in 30,35,74,92,103,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3) these positions.In certain embodiments, the amino acid that non-natural exists is connected at the certain position place with water-soluble polymers, described position includes, but is not limited to the position corresponding to one or more positions in 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3) these positions.In certain embodiments, the amino acid that non-natural exists is connected at the certain position place with water-soluble polymers, described position include, but is not limited to corresponding to 35,92,131,1 34,143,145 or these positions of its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3) in the position of one or more positions.In certain embodiments, the amino acid that non-natural exists is connected at the certain position place with water-soluble polymers, described position include, but is not limited to corresponding to 30,35,74,92,103,145 or these positions of its any combination (from SEQID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3) in the position of one or more positions.In certain embodiments, the amino acid that non-natural exists is connected at the certain position place with water-soluble polymers, described position include, but is not limited to corresponding to 35,92,143,145 or these positions of its any combination (from SEQ ID NO:2, SEQ ID NO:1 or 3 corresponding amino acid) in the position of one or more positions.In certain embodiments, amino acid that non-natural exists and water-soluble polymers are connected in the position corresponding to (but being not limited to) position 35 (from SEQID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).
In certain embodiments, (water-soluble polymers that for example, hGH) is connected comprises one or more peg molecules (PEG) with GH.Polymkeric substance (PEG) can be linear or branched for example.Usually, the linear polymer that is used for the present invention (for example, PEG) can have about 0.1kDa to the molecular weight of about 100kDa, or have the molecular weight that about 1kDa arrives about 60kDa, or have the molecular weight that about 20kDa arrives about 40kDa, or have the molecular weight of about 30kDa.Usually, the branched polymers that is used for the present invention (for example, PEG) can have about 1kDa to the molecular weight of about 100kDa, or have the molecular weight that about 30kDa arrives about 50kDa, or have the molecular weight of about 40kDa.Polymkeric substance such as PEG further describes in this article.In certain embodiments, (for example, hGH) (for example, the key between PEG) is the oxime key to GH with water-soluble polymers.
Some embodiment of the present invention is contained and is comprised that (for example, composition hGH), wherein said covalent linkage are the oxime keys to the GH that is connected with at least one water-soluble polymers by covalent linkage.In certain embodiments, water-soluble polymers is PEG, for example, and linear PEG.(for example containing by oxime key and GH, hGH) among some embodiment of the linear PEG of at least one that is connected, described PEG can have the molecular weight of about 0.1kDa to about 100kDa, or has about 1kDa to the molecular weight of about 60kDa, or have the molecular weight of about 20kDa, or has the molecular weight of about 30kDa to about 40kDa.(for example, among some embodiment of the linear PEG that hGH) is connected, described PEG has the molecular weight of about 30kDa by oxime key and GH containing.(for example containing by oxime key and GH, hGH) among some embodiment of at least one the branch PEG that is connected, described PEG can have the molecular weight of about 1kDa to about 100kDa, or has the molecular weight of about 30kDa to about 50kDa, or has the molecular weight of about 40kDa.(for example, among some embodiment of the branch PEG that hGH) is connected, described PEG has the molecular weight of about 40kDa by oxime key and GH containing.In certain embodiments, GH is the GH of hGH for example, and among some embodiment in these embodiments, GH (for example, hGH) have at least about 80% with the identical sequence of SEQ ID NO:2; In certain embodiments, GH (for example, hGH) has a sequence for the sequence of SEQ ID NO:2.In certain embodiments, GH (for example, hGH) contains at least one non-naturally encoded amino acid; Among some embodiment in these embodiments, at least one oxime key is between non-naturally encoded amino acid and at least one water-soluble polymers.In certain embodiments, non-naturally encoded amino acid contains carbonyl, such as, ketone group; In certain embodiments, non-naturally encoded amino acid is to acetylphenylalanine.In certain embodiments, be to replace to acetylphenylalanine in position corresponding to the position 35 of SEQ ID NO:2.
Therefore, in certain embodiments, the invention provides that a kind of (for example, (for example, hGH), wherein said covalent linkage is the oxime key to the GH that PEG) is connected by covalent linkage and at least one water-soluble polymers.In certain embodiments, water-soluble polymers is PEG, and described PEG is linear PEG.In these embodiments, linear PEG has the molecular weight of about 0.1kDa to about 100kDa, or has the molecular weight of about 1kDa to about 60kDa, or has the molecular weight of about 20kDa to about 40kDa, or has the molecular weight of about 30kDa.(for example, among some embodiment of the linear PEG that hGH) is connected, described PEG has the molecular weight of about 30kDa by oxime key and GH containing.In certain embodiments, water-soluble polymers is PEG, and described PEG is branch PEG.In these embodiments, branch PEG has the molecular weight of about 1kDa to about 100kDa, or has the molecular weight of about 30kDa to about 50kDa, or has the molecular weight of about 40kDa.(for example, among some embodiment of the branch PEG that hGH) is connected, described PEG has the molecular weight of about 40kDa by oxime key and GH containing.
In certain embodiments, (for example the invention provides a kind of GH, hGH), wherein said GH (for example, hGH) contain non-naturally encoded amino acid, wherein GH (for example, PEG) is connected by covalent linkage and at least one water-soluble polymers, and wherein said covalent linkage is non-naturally encoded amino acid and water-soluble polymers (for example, the oxime key between PEG).In certain embodiments, corresponding to the position of the position 35 of SEQ ID NO:2 non-naturally encoded amino acid is incorporated into GH (for example, hGH) in.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is linear PEG.In these embodiments, linear PEG has the molecular weight of about 0.1kDa to about 100kDa, or has the molecular weight of about 1kDa to about 60kDa, or has the molecular weight of about 20kDa to about 40kDa, or has the molecular weight of about 30kDa.(for example, among some embodiment of the linear PEG that hGH) is connected, described PEG has the molecular weight of about 30kDa by oxime key and GH containing.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is branch PEG.In these embodiments, branch PEG has the molecular weight of about 1kDa to about 100kDa, or has the molecular weight of about 30kDa to about 50kDa, or has the molecular weight of about 40kDa.(for example, among some embodiment of the branch PEG that hGH) is connected, described PEG has the molecular weight of about 40kDa by oxime key and GH containing.
In certain embodiments, (for example the invention provides a kind of GH, hGH), wherein said GH (for example, hGH) contain the promising non-naturally encoded amino acid whose non-naturally encoded amino acid that contains carbonyl, wherein GH (for example, PEG) is connected by covalent linkage and at least one water-soluble polymers, and described covalent linkage is the non-naturally encoded amino acid that contains carbonyl and water-soluble polymers (for example, the oxime key between PEG).In certain embodiments, corresponding to the position of the position 35 of SEQID NO:2 the non-naturally encoded amino acid that contains carbonyl is incorporated into GH (for example, hGH) in.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is linear PEG.In these embodiments, linear PEG has the molecular weight of about 0.1kDa to about 100kDa, or has the molecular weight of about 1kDa to about 60kD, or has the molecular weight of about 20kDa to about 40kDa, or has the molecular weight of about 30kDa.(for example, among some embodiment of the linear PEG that hGH) is connected, described PEG has the molecular weight of about 30kDa by oxime key and GH containing.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is branch PEG.In these embodiments, branch PEG has the molecular weight of about 1kDa to about 100kDa, or has the molecular weight of about 30kDa to about 50kDa, or has the molecular weight of about 40kDa.(for example, among some embodiment of the branch PEG that hGH) is connected, described PEG has the molecular weight of about 40kDa by oxime key and GH containing.
In certain embodiments, the invention provides a kind of contain comprise ketone group non-naturally encoded amino acid whose GH (for example, hGH), wherein said GH by covalent linkage and at least one water-soluble polymers (for example is, PEG) be connected, and wherein said covalent linkage is to contain the non-naturally encoded amino acid of ketone group and water-soluble polymers (for example, the oxime key between PEG).In certain embodiments, the non-naturally encoded amino acid that will contain ketone group corresponding to the position of the position 35 of SEQ ID NO:2 incorporate into GH (for example, hGH) in.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is linear PEG.In these embodiments, linear PEG has the molecular weight of about 0.1kDa to about 100kDa, or has the molecular weight of about 1kDa to about 60kD, or has the molecular weight of about 20kDa to about 40kDa, or has the molecular weight of about 30kDa.(for example, among some embodiment of the linear PEG that hGH) is connected, described PEG has the molecular weight of about 30kDa by oxime key and GH containing.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is branch PEG.In these embodiments, branch PEG has the molecular weight of about 1kDa to about 100kDa, or has the molecular weight of about 30kDa to about 50kDa, or has the molecular weight of about 40kDa.(for example, among some embodiment of the branch PEG that hGH) is connected, described PEG has the molecular weight of about 40kDa by oxime key and GH containing.
In certain embodiments, the invention provides and a kind ofly (for example contain promising non-naturally encoded amino acid whose GH to acetylphenylalanine, hGH), wherein said GH by covalent linkage and at least one water-soluble polymers (for example is, PEG) be connected, and wherein said covalent linkage is to acetylphenylalanine and water-soluble polymers (for example, the oxime key between PEG).In certain embodiments, will incorporate into acetylphenylalanine corresponding to the position of the position 35 of SEQ ID NO:2 GH (for example, hGH) in.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is linear PEG.In these embodiments, linear PEG has the molecular weight of about 0.1kDa to about 100kDa, or has the molecular weight of about 1kDa to about 60kD, or has the molecular weight of about 20kDa to about 40kDa, or has the molecular weight of about 30kDa.(for example, among some embodiment of the linear PEG that hGH) is connected, described PEG has the molecular weight of about 30kDa by oxime key and GH containing.Water-soluble polymers is among some embodiment of PEG therein, and described PEG is branch PEG.In these embodiments, branch PEG has the molecular weight of about 1kDa to about 100kDa, or has the molecular weight of about 30kDa to about 50kDa, or has the molecular weight of about 40kDa.(for example, among some embodiment of the branch PEG that hGH) is connected, PEG has the molecular weight of about 40kDa by oxime key and GH containing.
In certain embodiments, (for example the invention provides a kind of GH, hGH), it comprises SEQ ID NO:2, and wherein said GH (for example, hGH) be corresponding to the position of the position 35 of SEQ ID NO:2 through acetylphenylalanine is replaced, described is to be that the linear PEG of about 30kDa is connected by oxime key and molecular weight to acetylphenylalanine.
In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain at least one the non-naturally encoded amino acid that replaces in one or more positions, described position includes, but is not limited to corresponding to the position with upper/lower positions: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain at least one the non-naturally encoded amino acid that replaces in one or more positions, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 30,35,74,92,103,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain at least one the non-naturally encoded amino acid that replaces in one or more positions, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain at least one the non-naturally encoded amino acid that replaces in one or more positions, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 35,92,13 1,134,143,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain at least one the non-naturally encoded amino acid that replaces in one or more positions, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 30,35,74,92,103,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain at least one the non-naturally encoded amino acid that replaces in one or more positions, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 35,92,143,145 or its any combination (from SEQID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain at least one the non-naturally encoded amino acid that replaces in one or more positions, described position includes, but is not limited to the position corresponding to position 35 (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).At PEG is among the embodiment of linear PEG, and PEG can have the molecular weight of about 0.1kDa to about 100kDa, or has the molecular weight of about 1kDa to about 60kD, or has the molecular weight of about 20kDa to about 40kDa, or has the molecular weight of about 30kDa.
In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain replace in one or more positions at least one for to the non-naturally encoded amino acid of acetylphenylalanine, described position includes, but is not limited to corresponding to the position with upper/lower positions: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain replace in one or more positions at least one for to the non-naturally encoded amino acid of acetylphenylalanine, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 30,35,74,92,103,143,145 (SEQID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain replace in one or more positions at least one for to the non-naturally encoded amino acid of acetylphenylalanine, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain replace in one or more positions at least one for to the non-naturally encoded amino acid of acetylphenylalanine, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 35,92,131,134,143,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain replace in one or more positions at least one for to the non-naturally encoded amino acid of acetylphenylalanine, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 30,35,74,92,103,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQ ID NO:2, and wherein GH (for example, hGH) contain replace in one or more positions at least one for to the non-naturally encoded amino acid of acetylphenylalanine, described position includes, but is not limited to corresponding to the position with upper/lower positions: position 35,92,143,145 or its any combination (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the invention provides a kind of (for example comprising by oxime key and at least one PEG, linear PEG) GH that is connected (for example, hGH) hormonal composition, wherein said GH (for example, hGH) comprise the aminoacid sequence of SEQID NO:2, and wherein GH (for example, hGH) contain replace in one or more positions at least one for to the non-naturally encoded amino acid of acetylphenylalanine, described position includes, but is not limited to the position corresponding to position 35 (from SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).At PEG is among the embodiment of linear PEG, and PEG can have the molecular weight of about 0.1kDa to about 100kDa, or has the molecular weight of about 1kDa to about 60kD, or has the molecular weight of about 20kDa to about 40kDa, or has the molecular weight of about 30kDa.
In certain embodiments, (for example the invention provides a kind of GH, hGH), wherein said GH (for example, hGH) contain at least one non-naturally encoded amino acid, wherein GH is connected by covalent linkage and a plurality of water-soluble polymers (for example, a plurality of PEG), one of them or above covalent linkage are at least one non-naturally encoded amino acid and water-soluble polymers (for example, the oxime key between PEG).GH (for example, hGH) can with about 2-100 water-soluble polymers (for example, PEG) be connected, or with about 2-50 water-soluble polymers (for example, PEG) be connected, or with about 2-25 water-soluble polymers (for example, PEG) be connected, or with about 2-10 water-soluble polymers (for example, PEG) be connected, or with about 2-5 water-soluble polymers (for example, PEG) be connected, or (for example, PEG) be connected with about 5-100 water-soluble polymers, or with about 5-50 water-soluble polymers (for example, PEG) be connected, or (for example, PEG) be connected with about 5-25 water-soluble polymers, or with about 5-10 water-soluble polymers (for example, PEG) be connected, or (for example, PEG) be connected with about 10-100 water-soluble polymers, or with about 10-50 water-soluble polymers (for example, PEG) be connected, or (for example, PEG) be connected with about 10-20 water-soluble polymers, or with about 20-100 water-soluble polymers (for example, PEG) be connected, or (for example, PEG) be connected with about 20-50 water-soluble polymers, or (for example, PEG) be connected with about 50-100 water-soluble polymers.Can any position as herein described with one or more non-naturally encoded amino acid incorporate into GH (for example, hGH) in.In certain embodiments, corresponding to the position of the position 35 of SEQ ID NO:2 with at least one non-naturally encoded amino acid incorporate into GH (for example, hGH) in.In certain embodiments, non-naturally encoded amino acid comprises that at least one is the non-naturally encoded amino acid whose non-naturally encoded amino acid that contains carbonyl, for example, contains the non-naturally encoded amino acid of ketone group, such as, to acetylphenylalanine.In certain embodiments, GH (for example, hGH) comprises acetylphenylalanine.In certain embodiments, corresponding to the position of the position 35 of SEQ ID NO:2 will to acetylphenylalanine incorporate into GH (for example, hGH) in, be to be connected wherein by one in oxime key and the polymkeric substance (for example, among the PEG) to acetylphenylalanine.In certain embodiments, at least one water-soluble polymers (PEG) is by (for example, hGH) being connected with GH with at least one non-naturally encoded amino acid whose covalent linkage for example.In certain embodiments, described covalent linkage is the oxime key.In certain embodiments, a plurality of water-soluble polymerss (PEG) are by (for example, hGH) being connected with GH with a plurality of non-naturally encoded amino acid whose covalent linkage for example.In certain embodiments, at least one covalent linkage is the oxime key; In certain embodiments, a plurality of covalent linkage are the oxime key; In certain embodiments, all substantially keys are the oxime key.A plurality of water-soluble polymerss (for example, PEG) can be linear polymer, branched polymers or its any combination.In incorporating the embodiment of one or more linear PEG into, linear PEG has the molecular weight of about 0.1kDa to about 100kDa, or have the molecular weight of about 1kDa to about 60kDa, or have the molecular weight of about 20kDa to about 40kDa, or have the molecular weight of about 30kDa.In the embodiment that incorporates one or more branches PEG into, branch PEG has the molecular weight of about 1kDa to about 100kDa, or has the molecular weight of about 30kDa to about 50kDa, or has the molecular weight of about 40kDa.Should be appreciated that (for example, embodiment PEG) generally speaking will adopt molecular weight ratio to use the low described polymkeric substance of embodiment of single PEG to adopt a plurality of water-soluble polymerss.Thereby in certain embodiments, the total molecular weight of a plurality of PEG is about 0.1-500kDa, or be about 0.1-200kDa, or be about 0.1-100kDa, or be about 1-1000kDa, or be about 1-500kDa, or be about 1-200kDa, or be about 1-100kDa, or be about 10-1000kDa, or be about 10-500kDa, or be about 10-200kDa, or be about 10-100kDa, or be about 10-50kDa, or be about 20-1000kDa, or be about 20-500kDa, or be about 20-200kDa, or be about 20-100kDa, or be about 20-80kDa, and be about 20-60kDa, be about 5-100kDa, be about 5-50kDa, or be about 5-20kDa.
Human GH antagonist includes, but is not limited in the position 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 and 127 places to have and replaces or 1 place has interpolation (promptly in the position, at N end) or its any combination (SEQ ID NO:2, or SEQ ID NO:1,3 or any other GH sequence in corresponding amino acid) those antagonists.
Can with various non-naturally encoded aminoacid replacement to or incorporate GH (for example, the hGH) specified location in the polypeptide into.Generally speaking, based on GH (for example, hGH) research of the three-dimensional crystalline structure of polypeptide and its acceptor, conservative property replaces preferred (promptly, non-naturally encoded amino acid based on aryl, such as acetylphenylalanine or O-propargyl tyrosine are replaced Phe, Tyr or Trp) and the those skilled in the art (for example wish to introduce GH, hGH) specificity in the polypeptide in conjunction with chemical action (for example, if the those skilled in the art want to realize with Hu Shi root [3+2] cycloaddition of water-soluble polymers with alkynyl moiety or with the amido linkage formation effect of water-soluble polymers that has and incorporate into the aryl ester of phosphine part, so just introduce 4-triazobenzene L-Ala), select the specific non-naturally encoded amino acid that is suitable for incorporating into.
In one embodiment, method comprises in addition: non-natural amino acid is incorporated in the protein, and wherein said non-natural amino acid comprises first reactive group; With make protein and the molecule that comprises second reactive group (include, but is not limited to mark, dyestuff, polymkeric substance, water-soluble polymers, the derivative of polyoxyethylene glycol, the photocrosslinking agent, radionuclide, cytotoxin compounds, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, the antisense polynucleotide, carbohydrate, water-soluble branch-shape polymer, cyclodextrin, inhibition Yeast Nucleic Acid, biomaterial, nanoparticle, spin labeling, fluorophore, metallic part, radioactive segment, novel functional group, covalently or non-covalentlyly with the group of other interaction of molecules, light cage latching segment, but actinic radiation excitation portion, the intramolecular photosensitization part, vitamin H, the derivative of vitamin H, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, the side chain of elongation, sugar through the carbon connection, redox active agent, amino thioic acid sulfoacid, toxic moiety, through isotope-labeled part, the biophysics probe, phosphorescent group, chemiluminescent groups, the intensive group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable mark, small molecules, quantum dot, the nanometer transmitter, the radioactive nuleus thuja acid, the radioactivity transmitter, any combination or any other of neutron capture agent or above-mentioned substance are wanted compound or material) contact.Described first reactive group and described second reaction-ity group reaction are to make molecule be connected with non-natural amino acid by [3+2] cycloaddition.In one embodiment, first reactive group is alkynyl or azido-part, and second reactive group is azido-or alkynyl part.For example, first reactive group is alkynyl part (includes, but is not limited to non-natural amino acid to the propargyloxy phenylalanine in), and second reactive group is the azido-part.In another example, first reactive group be azido-part (include, but is not limited to non-natural amino acid right-azido--L-phenylalanine in), and second reactive group is the alkynyl part.
In some cases, (for example, hGH) other interpolation in the polypeptide, replacement or disappearance combination are to influence GH (for example, hGH) other biological nature of polypeptide with making non-naturally encoded aminoacid replacement and GH.In some cases, other interpolation, replacement or disappearance can increase GH (for example, hGH) stability of polypeptide (including, but is not limited to resistance) to proteolytic degradation or increase GH (for example, hGH) polypeptide to the avidity of its acceptor.In certain embodiments, (for example, hGH) polypeptide comprises the aminoacid replacement that is selected from the group that is made up of following each replacement to GH: the F10A among the SEQID NO:2, F10H, F10I; M14W, M14Q, M14G; H18D; H21N; G120A; R167N; D171S; E174S; F176Y, I179T or its any combination.In some cases, other interpolation, replacement or disappearance can increase GH (for example, the hGH) solvability of polypeptide (including, but is not limited to when expressing) in intestinal bacteria or other host cell.In certain embodiments, add, replace or lack and to be increased in the polypeptide solvability after the expression in intestinal bacteria or other recombinant host cell.In certain embodiments, except that the non-natural amino acid whose position that the polypeptide solvability that is suitable for incorporating into after causing in intestinal bacteria or other recombinant host cell expressing increases, also select to be suitable for other position that replaces with amino acid natural coding or non-natural.In certain embodiments, GH (for example, hGH) polypeptide comprises another interpolation, replacement or disappearance, and its regulation and control are to GH (for example, the hGH) avidity of polypeptide receptor, regulation and control (including, but is not limited to strengthen or weaken) receptor dimerizationization, stablize receptor dimer, the regulation and control circulating half-life, regulation and control discharge or bioavailability, promote purifying, or improve or change specific dosing way.For example, except that introducing one or more non-naturally encoded amino acid as described herein, also introduce one or more following replace increase GH (for example, hGH) varient to the avidity of its acceptor: F10A, F10H or F10I; M14W, M14Q or M14G; H18D; H21N; R167N; D171S; E174S; F176Y and I179T.Similarly, GH (for example, hGH) polypeptide can comprise chemistry or enzymatic lysis sequence, protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or polyhistidyl) or other sequence based on avidity (including, but is not limited to FLAG, polyhistidyl, GST etc.) or link molecule (including, but is not limited to vitamin H), and it improve to detect (including, but is not limited to GFP), purifying, reduces or other characteristic of polypeptide by transportation, prodrug release or activation, the hGH size of tissue or cytolemma.
In certain embodiments, non-naturally encoded amino acid whose replacement produces GH (for example, hGH) antagonist.The subclass that is suitable for incorporating into one or more non-naturally encoded amino acid whose exemplary positions comprises: the interpolation before position 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123,127 or the position 1 (SEQ ID NO:2, SEQ ID NO:1,3 or the corresponding amino acid of any other GH sequence).In certain embodiments, GH (for example, hGH) antagonist comprise make GH serve as antagonist with at least one replacement in the lower area: 1-5 (N end), 6-33 (spiral A), the 34-74 (zone between spiral A and the spiral B, the A-B ring), 75-96 (spiral B), the 97-105 (zone between spiral B and the spiral C, the B-C ring), 106-129 (spiral C), 130-153 (zone between spiral C and the spiral D, C-D ring), 154-183 (spiral D), 184-191 (C end).In other embodiments, incorporate aminoterminal zone and the interior residue of a part of spiral C that non-naturally encoded amino acid whose exemplary position is included in spiral A into.In another embodiment, with non-naturally encoded aminoacid replacement G120 such as right-azido--L-phenylalanine or O-propargyl-L-tyrosine.In other embodiments, the replacement listed above and other are replaced combination, make that (for example, hGH) polypeptide becomes GH (for example, hGH) antagonist to GH.For example, a position in the position of being differentiated is with non-naturally encoded aminoacid replacement in this article, and introduces replacement (for example, G120R, G120K, G120W, G120Y, G120F or G120E) at G120 place simultaneously.In certain embodiments, GH (for example, hGH) antagonist comprise be connected with water-soluble polymers be present in GH (for example, hGH) the non-naturally encoded amino acid in the molecular receptor calmodulin binding domain CaM.
In some cases, 1,2,3,4,5,6,7,8,9, the amino acid more than 10 or 10 are through one or more non-naturally encoded aminoacid replacement.In some cases, (for example, hGH) polypeptide comprises that in addition 1,2,3,4,5,6,7,8,9, one or more the non-naturally encoded amino acid more than 10 or 10 are to naturally occurring amino acid whose replacement to GH.For example, in certain embodiments, GH (for example, hGH) with one or more residues in the lower area through one or more non-naturally encoded aminoacid replacement: 1-5 (N end); 32-46 (the N end of A-B ring); 97-105 (B-C ring); And 132-149 (C-D ring); And 184-191 (C end).In certain embodiments, GH (for example, hGH) with one or more residues in the lower area through one or more non-naturally encoded aminoacid replacement: 1-5 (N end), 6-33 (spiral A), the 34-74 (zone between spiral A and the spiral B, the A-B ring), 75-96 (spiral B), the 97-105 (zone between spiral B and the spiral C, the B-C ring), 106-129 (spiral C), 130-153 (zone between spiral C and the spiral D, C-D ring), 154-183 (spiral D), 184-191 (C end).In some cases, one or more non-naturally encoded residues are to be connected with the linearity or the branch PEG (the about 5-20kDa of quality or littler) of one or more lower molecular weights, and then with respect to the material that is connected with PEG single, higher molecular weight, its binding affinity is strengthened and serum half-life suitable.
In certain embodiments, following GH (for example, in residue hGH) at the most two residues in following position through one or more non-naturally encoded aminoacid replacement: position 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187.In some cases, carry out any replacement in the following paired replacement: K38X *And K140X *K41X *And K145X *Y35X *And E88X *Y35X *And F92X *Y35X *And Y143X *F92X *And Y143X *, X wherein *Represent non-naturally encoded amino acid.Be suitable for incorporating into the combination that two or more non-naturally encoded amino acid whose preferred sites comprise following residue: position 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187.Be suitable for incorporating into the combination that two or more non-naturally encoded amino acid whose particularly preferred positions comprise following residue: position 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155.
Be suitable for the amino acid that two or more are non-naturally encoded and (for example incorporate GH into, hGH) preferred site in comprises the combination of following residue: from before the position 1 of SEQ ID NO:2 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (that is, at proteinic carboxyl terminal) or its any combinations.
VII. the expression in non-eukaryote and eukaryote
For (for example obtaining clone GH, hGH) high level expression of polynucleotide, the those skilled in the art (for example will encode GH of the present invention usually, hGH) the polynucleotide subclone of polypeptide is in expression vector, described expression vector contains and instructs the strong promoter transcribe, transcribes/translation termination, and if for the nucleic acid of coded protein, then it also contains the ribosome binding site that is useful on translation initiation.Suitable bacterium promotor is known to the those skilled in the art and describes (for example) in philtrums such as people such as Sambrook and Ausubel.
(for example be used to express GH of the present invention, hGH) bacterial expression system of polypeptide can be at (including, but is not limited to) intestinal bacteria, bacillus kind (Bacillus sp.), Pseudomonas fluorescens, Pseudomonas aeruginosa, pseudomonas putida and salmonella (Salmonella)) in obtain (people such as Palva, Gene:22:229-235 (1983); People such as Mosbach, Nature 302:543-545 (1983)).The test kit that is used for described expression system can be buied.The eukaryotic expression system that is used for mammalian cell, yeast and insect cell is known to the those skilled in the art and also is to buy.(for example, hGH) under the situation of polypeptide, the ability that is based on its use quadrature component for the host cell of expression is selected using quadrature tRNA and aminoacyl tRNA synthetase (above institute describes) to express GH of the present invention.Exemplary host cell comprises gram positive bacterium (Gram-positive bacteria) (including, but is not limited to bacillus pumilus (B.brevis), subtilis (B.subtilis) or streptomyces (Streptomyces)) and gram negative bacterium (Gram-negative bacteria) (intestinal bacteria, Pseudomonas fluorescens, Pseudomonas aeruginosa, pseudomonas putida bacterium) and yeast and other eukaryotic cell.Can use as described herein and comprise the right cell of O-tRNA/O-RS.
Eukaryotic host cell of the present invention or non-eukaryotic host cell provide the synthetic proteinic ability that comprises useful in a large number alpha-non-natural amino acid.On the one hand, composition optionally comprises (including, but is not limited to) at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, restrains at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 or the above protein that comprises alpha-non-natural amino acid of 1 gram, or comprises the amount that available in vivo protein production method (detail file of relevant recombinant protein generation and purifying are provided) obtains herein.On the other hand, described protein optionally is to include, but is not limited to cellular lysate, damping fluid, include, but is not limited to whenever rise to the protein of few 10 micrograms in medicine damping fluid or other liquid suspension (including, but is not limited to) for including, but is not limited to about 1nl to any amount of volume between about 100L, whenever rise to the protein of few 50 micrograms, whenever rise to the protein of few 75 micrograms, whenever rise to the protein of few 100 micrograms, whenever rise to the protein of few 200 micrograms, whenever rise to the protein of few 250 micrograms, whenever rise to the protein of few 500 micrograms, whenever rise to few 1 milligram protein or whenever rise to few proteinic concentration more than 10 milligrams or 10 milligrams and exist in the composition.The protein that comprises at least one alpha-non-natural amino acid that produces a large amount of (including, but is not limited to, may be with the big amount of the amount that other method was produced that includes, but is not limited in vitro translate than usually) in eukaryotic cell is a feature of the present invention.
Eukaryotic host cell of the present invention or non-eukaryotic host cell provide biosynthesizing to comprise the proteinic ability of useful in a large number alpha-non-natural amino acid.For example, comprising the protein of alpha-non-natural amino acid can be at cell extract, cellular lysate, substratum, following concentration in the damping fluid etc. produces: include, but is not limited at least 10 micrograms per litre, at least 50 micrograms per litre, at least 75 micrograms per litre, at least 100 micrograms per litre, at least 200 micrograms per litre, at least 250 micrograms per litre or at least 500 micrograms per litre, at least 1 mg/litre, at least 2 mg/litre, at least 3 mg/litre, at least 4 mg/litre, at least 5 mg/litre, at least 6 mg/litre, at least 7 mg/litre, at least 8 mg/litre, at least 9 mg/litre, at least 10 mg/litre, at least 20 mg/litre, at least 30 mg/litre, at least 40 mg/litre, at least 50 mg/litre, at least 60 mg/litre, at least 70 mg/litre, at least 80 mg/litre, at least 90 mg/litre, at least 100 mg/litre, at least 200 mg/litre, at least 300 mg/litre, at least 400 mg/litre, at least 500 mg/litre, at least 600 mg/litre, at least 700 mg/litre, at least 800 mg/litre, at least 900 mg/litre, 1 grams per liter, 5 grams per liters, the protein that 10 grams per liters or 10 grams per liters are above.
I. Expression system, cultivation and separate
GH (for example, hGH) can express in the many appropriate expression system that comprise (for example) yeast, insect cell, mammalian cell and bacterium by polypeptide.The description of exemplary expression system is provided in hereinafter.
YeastAs used herein term " yeast " comprises can express coding GH (for example, hGH) any yeast in each primary yeast of the gene of polypeptide.Described yeast includes, but is not limited to ascosporogenous yeast (Endomycetale (Endomycetales)), product basidiospore yeast (basidiosporogenous yeast) and belongs to the yeast of fungi imperfecti (blastogenesis Zoopagales (Blastomycetes)) group.Ascosporogenous yeast is divided into 2 sections, Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter comprises 4 subfamilies, Schizosaccharomycoideae (Schizosaccharomycoideae) (for example, Schizosaccharomyces (genus Schizosaccharomyces)), Nadsonioideae (Nadsonioideae), Lipomycetoideae (Lipomycoideae) and yeast subfamily (Saccharomycoideae) (for example, pichia belongs to (genera Pichia), kluyveromyces spp (Kluyveromyces) and Saccharomycodes (Saccharomyces)).Producing the basidiospore yeast comprises basidiomycetes Leucosporidium (genera Leucosporidium), Rhodosporidium (Rhodosporidium), locks and throw yeast belong (Sporidiobolus), Filobasidiella (Filobasidium) and incense ashes plan lock load Pseudomonas (Filobasidiella).The yeast that belongs to fungi imperfecti (blastogenesis Zoopagales) group is divided into 2 sections, Sporobolomycetaceae (Sporobolomycetaceae) (for example, Sporobolomyces (genera Sporobolomyces) and cloth are reined in and are played spore yeast belong (Bullera)) and Cryptococcaceae (Cryptococcaceae) (for example, Candida (genus Candida)).
For paying close attention to especially of using of the present invention is species in the following species: pichia belongs to, kluyveromyces spp, Saccharomycodes, Schizosaccharomyces, Hansenula (Hansenula), torulopsis (Torulopsis) and Candida include, but is not limited to pichia pastoris phaff (P.pastoris), turn round and look at Le Modi pichia spp (P.guillerimondii), yeast saccharomyces cerevisiae (S.cerevisiae), Ka Ersibai yeast (S.carlsbergensis), saccharomyces diastaticus (S.diastaticus), Douglas yeast (S.douglasii), Crewe Wei Er yeast (S.kluyveri), Nuo Bensi yeast (S.norbensis), ellipsoideus yeast (S.oviformis), Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis), Candida albicans (C.albicans), maltose candiyeast (C.maltosa) and many types of debaryomyces hansenii (H.polymorpha).
Selection is used to express GH, and (for example, hGH) suitable yeast of polypeptide is to belong in those skilled in the art's the skill.When selecting the yeast host that is used to express, appropriate host can comprise that demonstration has (for example) good secretion capacity, low proteolytic activity, good secretion capacity, good soluble protein produces and those hosts of whole robustness.Yeast generally can obtain from the various sources that include, but is not limited to following source: Yeast GeneticStock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA) and American Type Culture Collection (" ATCC ") (Manassas, VA).
Term " yeast host " or " yeast host cell " comprise the yeast that can be used as or be used as the recipient of recombinant vectors or other transfer DNA.Described term comprises the filial generation of the original yeast host cell of accepting recombinant vectors or other transfer DNA.Should be appreciated that because sudden change accidental or that have a mind to, the filial generation of single parental cell is aspect morphology or can be identical aspect original parental generation complementary genome DNA or total DNA.With treat by (for example, hGH) filial generation of the quite similar parental cell of the parental generation that characterizes of the relevant nature of the existence of the nucleotide sequence of polypeptide is included in filial generation of this definition indication such as coding GH.
Develop the expression and the conversion carrier that comprise extrachromosomal replication or integrative vector and be used for being transformed into many yeast hosts.For example, developed expression vector (people such as Sikorski, GENETICS (1989) 122:19 that is used for yeast saccharomyces cerevisiae; People such as Ito, J.BACTERIOL. (1983) 153:163; People such as Hinnen, PROC.NATL.ACAD.SCI.USA (1978) 75:1929); The expression vector (people such as Kurtz, the M that are used for Candida albicans OL.CELL.BIOL. (1986) 6:142); The expression vector (people such as Kunze, the J.BASIC M that are used for the maltose candiyeast ICROBIOL. (1985) 25:141); The expression vector (people such as Gleeson, the J.G that are used for many types of debaryomyces hansenii EN.M ICROBIOL. (1986) 132:3459; People such as Roggenkamp, MOL.GENETICS AND GENOMICS (1986) 202:302); The expression vector (people such as Das, J.BACTERIOL. (1984) 158:1165) that is used for Kluyveromyces fragilis; The expression vector (people such as De Louvencourt, J.BACTERIOL. (1983) 154:737 that are used for Kluyveromyces lactis; People such as Van denBerg, BIOTECHNOLOGY (NY) (1990) 8:135); The expression vector (people such as Kunze, the J.BASIC M that are used for the Le Modi pichia spp ICROBIOL. (1985) 25:141); Be used for expression vector (United States Patent (USP) the 5th, 324, No. 639, the 4th, 929, No. 555 and the 4th, 837, No. 148 of pichia pastoris phaff; People such as Gregg, M OL.C ELL.B IOL. (1985) 5:3376); The expression vector (people such as Beach, NATURE (1982) 300:706) that is used for schizosaccharomyces pombe (Schizosaccharomyces pombe); With the expression vector that is used to separate fat Ye Luoweiya yeast (Y.lipolytica); The expression vector (people such as Ballance, the B that are used for Aspergillus nidulans (A.nidulans) IOCHEM.B IOPHYS.R ES.C OMMUN. (1983) 112:284-89; People such as Tilburn, G ENE(1983) 26:205-221; With people such as Yelton, PROC.N ATL.A CAD.S CI.USA (1984) 81:1470-74); The expression vector (Kelly and Hynes, EMBO J. (1985) 4:475-479) that is used for melanomyces (A.niger); The expression vector (EP 0 244 234) that is used for Trichodermareesei (T.reesia); With the expression vector (WO 91/00357) that is used for such as the filamentous fungus of Neurospora (Neurospora), Penicillium (Penicillium), the curved mould genus of neck (Tolypocladium), each reference is to be incorporated herein by reference.
The control sequence of yeast vector is known to the those skilled in the art, and includes, but is not limited to from the promoter region such as the gene of following gene: alcoholdehydrogenase (ADH) (EP 0 284 044); Enolase; Glucokinase; The G-6-P isomerase; Glyceraldehyde-3-phosphate-desaturase (GAP or GAPDH); Hexokinase; Phosphofructokinase; The 3-phoshoglyceric acid mutase; And pyruvate kinase (PyK) (EP 0 329 203).The yeast PHO5 gene of coding acid phosphatase also can provide promoter sequence (people such as Miyanohara, the PROC.N of usefulness ATL.A CAD.SCI.USA (1983) 80:1).Other the suitable promoter sequence that uses for yeast host can comprise glycerol 3-phosphate acid kinase (people such as Hitzeman, J.BIOL.C HEM. (1980) 255:12073) and such as other glycolytic ferment (people such as Holland, BIOCHEMISTRY (1978) 17:4900 of pyruvic carboxylase, triosephosphate isomerase and phosphoglucose isomerase; People such as Hess, J.A DV.E NZYMER EG. (1969) 7:149) promotor.Can comprise the promoter region of the enzyme of alcoholdehydrogenase 2, different cell pigment C, acid phosphatase, metallothionein(MT), glyceraldehyde-3-phosphate dehydrogenase, the degrading enzyme relevant and responsible maltose and galactose utilization for transcribe induced Yeast promoter by growth conditions control with nitrogen metabolism with additional advantage.The suitable carriers and the promotor that are applicable to yeast expression are further described among the EP 0 073 657.
The yeast enhanser also can use with Yeast promoter.In addition, the synthetic promotor also can play Yeast promoter.For example, the upstream activating sequence of Yeast promoter (UAS) can engage with the transcriptional activation zone of another Yeast promoter, produces the synthetic hybrid promoters.The example of described hybrid promoters comprises the ADH regulating and controlling sequence that is connected with GAP transcriptional activation zone.Referring to, United States Patent (USP) the 4th, 880, No. 734 and the 4th, 876, No. 197, it is to be incorporated herein by reference.Other example of hybrid promoters comprise by with the promotor of forming such as the regulating and controlling sequence of ADH2, GAL4, GAL10 or the PHO5 gene of the combination of the glycolytic ferment gene transcription active region of GAP or PyK.Referring to, EP 0 164 556.In addition, Yeast promoter can comprise the naturally occurring promotor in the non-yeast source with ability that combining yeast RNA polymerase and initiation transcribe.
Other controlling elements that can comprise the part Yeast expression carrier comprises (for example) terminator (people such as Holland, J.BIOL.CHEM. (1981) 256:1385) from GAPDH or enolase gene.In addition, the replication orgin from 2 μ plasmid-deriveds is applicable to yeast.Suitable selection gene is the trp1 gene that is present in the yeast plasmid to be applicable to zymic.Referring to, people such as Tschumper, GENE (1980) 10:157; People such as Kingsman, GENE (1979) 7:141.The trp1 gene provides selectable marker for the yeast mutation bacterial strain that can't grow in tryptophane.Similarly, the yeast strain (ATCC 20,622 or 38,626) of scarce Leu2 is to replenish by the known plasmid that has the Leu2 gene.
The method that exogenous DNA is introduced in the yeast host is known to the those skilled in the art, and generally includes the complete yeast host cell that (but being not limited to) handle with alkaline kation or the conversion of spheroplast.For example, zymic transforms can be according to people such as Hsiao, P ROC.N ATL.A CAD.SCI.USA people such as (1979) 76:3829 and Van Solingen, J.B ACT. the method described in (1977) 130:946 is carried out.Yet such as being used for of merging by nuclear injection, electroporation or protoplastis DNA being introduced other method of cell also can be as people such as SAMBROOK, MOLECULAR CLONING:A L AB.MANUAL general described use the in (2001).Then can use the known standard technique culturing yeast of those skilled in the art host cell.
Being used in proteinic other method of yeast host cell expressing heterologous is known to the those skilled in the art.Generally referring to, No. the 20020055169th, U.S. Patent Publication case, No. the 6th, 361,969, United States Patent (USP); The 6th, 312, No. 923; The 6th, 183, No. 985; The 6th, 083, No. 723; The 6th, 017, No. 731; The 5th, 674, No. 706; The 5th, 629, No. 203; The 5th, 602, No. 034 and the 5th, 089, No. 398; Patent RE37, No. 343 and RE35, No. 749 are reviewed by the U.S.; PCT publication 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 and EP 0 164 556.Also referring to people such as Gellissen, A NTONIEV ANL EEUWENHOEK(1992) 62 (1-2): 79-93; People such as Romanos, YEAST (1992) 8 (6): 423-488; Goeddel, METHODS IN ENZYMOLOGY (1990) 185:3-7, each reference is to be incorporated herein by reference.
The yeast host bacterial strain can use the known standard fed-batch fermentation of those skilled in the art method to grow in the fermentor tank in the amplification stage.Because the carbon of specific yeast host utilizes the path or expresses the difference of the pattern of control, can be adjusted fermentation process.For example, the fermentation of Saccharomycodes yeast host may need single glucose charging, compound nitrogen source (for example, caseic hydrolysate) and vitamin supplement repeatedly.By contrast, the methylotrophic yeast pichia pastoris phaff may need glycerine, methyl alcohol and trace minerals charging, but only needs simple ammonium (nitrogen) salt to obtain optimum growh and expression.Referring to, for example, United States Patent (USP) the 5th, 324, No. 639; People such as Elliott, J.PROTEIN CHEM. (1990) 9:95; With people such as Fieschko, BIOTECH.BIOENG. (1987) 29:1113, it is to be incorporated herein by reference.
Yet, being independent of the yeast host bacterial strain that is adopted, described fermentation process can have some common characteristic.For example, can the growth limitation nutrition that be generally carbon be added in the fermentor tank to allow maximum growth in the amplification stage.In addition, fermentation process generally adopts the fermention medium of the carbon, nitrogen, basic salt, phosphorus and other trace nutrient (VITAMIN, trace minerals and salt etc.) that contain q.s through design.The example that is suitable for the fermention medium that uses for pichia is to be described in United States Patent (USP) the 5th, 324, and No. 639 and the 5th, 231, in No. 178, described patent is incorporated herein by reference.
Insect cell through baculovirus infectionTerm " insect host " or " insect host cell " refer to the insect that can be used as or be used as the recipient of recombinant vectors or other transfer DNA.Described term comprises the filial generation of transfected protentomon host cell.Should be appreciated that because sudden change accidental or that have a mind to, the filial generation of single parental cell is aspect morphology or can be identical aspect original parental generation complementary genome DNA or total DNA.With treat by (for example, hGH) filial generation of the quite similar parental cell of the parental generation that characterizes of the relevant nature of the existence of the nucleotide sequence of polypeptide is included in filial generation of this definition indication such as coding GH.
(for example, hGH) selection of the suitable insect cell of polypeptide is known to the those skilled in the art to be used to express GH.Some insect species are described fully in this technology and can be buied, and comprise Aedes aegypti (Aedes aegypti), silkworm (Bombyx mori), drosophila melanogaster (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and cabbage looper (Trichoplusia ni).When selecting the insect host that is used to express, appropriate host can comprise that demonstration especially has good secretion capacity, low proteolytic is active and those hosts of whole robustness.Insect generally can obtain from the various sources that include, but is not limited to following source: Insect GeneticStock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); With American Type Culture Collection (" ATCC ") (Manassas, VA).
Usually, comprise transfer vector, be generally bacterial plasmid, the suitable restriction site that it contains the genomic fragment of baculovirus and is used to insert heterologous gene to be expressed through the component of the insect expression system of baculovirus infection; Have with transfer vector in the wild-type baculovirus (this allow heterologous gene the homologous recombination in the baculovirus genome) of baculovirus specific fragment homologous sequence; With suitable insect host cell and growth medium.Being used for carrier construction, transfectional cell, picking plaque, making cell grow in the medium material of culture, method and technology is obtainable in this technology for handbook known and that describe these technology.
After inserting heterologous gene in the transfer vector, carrier and wild-type virus genome are transfected in insect host cell, in carrier described in the described host cell and viral genome reorganization.Expression is through recombinant virus and the discriminating and the purification of Recombinant plaque of packing.The material and the method that are used for baculovirus/insect cell expression system are so that (Carlsbad, kit form CA) is buied from (for example) Invitrogen Corp..These technology are generally known to the those skilled in the art and at SUMMERS that incorporates this paper by reference into and SMITH, have carried out comprehensive description among the TEXAS AGRICULTURALEXPERIMENT STATION BULLETIN NO.1555 (1987).Also referring to, RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY:BACULOVIRUSEXPRESSION PROTOCOLS (1995); People such as AUSUBEL, CURRENT PROTOCOLS INMOLECULAR BIOLOGY 16.9-16.11 (1994); K INGAnd P OSSEE, THE BACULOVIRUSSYSTEM:A LABORATORY GUIDE (1992); And O ' R EILLYDeng the people, BACULOVIRUSEXPRESSION VECTORS:A LABORATORY MANUAL (1992).
In fact, using the generation of the various heterologous proteins of baculovirus/insect cell expression system is known to the those skilled in the art.Referring to, for example, United States Patent (USP) the 6th, 368, No. 825; The 6th, 342, No. 216; The 6th, 338, No. 846; The 6th, 261, No. 805; The 6th, 245, No. 528; The 6th, 225, No. 060; The 6th, 183, No. 987; The 6th, 168, No. 932; The 6th, 126, No. 944; The 6th, 096, No. 304; The 6th, 013, No. 433; The 5th, 965, No. 393; The 5th, 939, No. 285; The 5th, 891, No. 676; The 5th, 871, No. 986; The 5th, 861, No. 279; The 5th, 858, No. 368; The 5th, 843, No. 733; The 5th, 762, No. 939; The 5th, 753, No. 220; The 5th, 605, No. 827; The 5th, 583, No. 023; The 5th, 571, No. 709; The 5th, 516, No. 657; The 5th, 290, No. 686; WO 02/06305; WO 01/90390; WO 01/27301; WO 01/05956; WO 00/55345; 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, and described reference is to be incorporated herein by reference.
The carrier that is applicable to baculovirus/insect cell expression system is known in this technology and comprises that (for example) is derived to the insect of baculovirus autographa california (Autographacalifornica) nuclear polyhedrosis virus (AcNPV) of the virus expression carrier that do not rely on helper virus (helper) and express and transfer vector.The virus expression carrier that is derived from this system generally uses the strong virus polyhedrin gene promoter to impel expression of heterologous genes.Generally referring to people such as O ' Reilly, BACULOVIRUS EXPRESSION VECTORS:A LABORATORYMANUAL (1992).
Before foreign gene being inserted in the shaft-like viral genome, dislocation construction (transfer vector) in the middle of the said components that will comprise promotor, leader sequence (time) if necessary, the encoding sequence of being paid close attention to and transcription termination sequence usually is assembled into.The construction that often centre misplaced maintain such as can stable maintenance in replicon such as the extra-chromosomal element among the host of bacterium (for example, plasmid).Replicon will have dubbing system, therefore allows it to be maintained the suitable host of cloning and increasing being used for.More particularly, plasmid can contain polyhedrin polyadenylation signal (Miller, ANN.REV.MICROBIOL. (1988) 42:177) and be used for selecting and prokaryotic organism penbritin (ampicillin) resistance (amp) gene and the replication orgin of breeding intestinal bacteria.
The transfer vector a kind of commonly used that is used for foreign gene is introduced AcNPV is pAc373.Also designed many other for the carrier known to the those skilled in the art, comprise (for example) pVL985, it changes over ATT with the polyhedrin initiator codon from ATG, and introduces BamHI clone positions at 32 base pair places in ATT downstream.Referring to Luckow and Summers, VIROLOGY 170:31 (1989).Other carrier that can buy comprise (for example) PBlueBac4.5/V5-His, pBlueBacHis2, pMelBac, pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
After inserting heterologous gene, with transfer vector and wild-type baculovirus genome cotransfection in the insect cell host.The method that is used for allogeneic dna sequence DNA is introduced the position of wanting of baculovirus is known in this technology.Referring to, S UMMERSAnd S MITH, T EXASA GRICULTURALE XPERIMENTS TATIONB ULLETINNo.1555 (1987); People such as Smith, MOL.CELL.BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989) 170:31.For example, insert to can be and be inserted in the gene such as polyhedron gene by homology double exchange reorganization; Insert also to can be and be inserted in designing the restriction enzyme position in desired baculovirus gene.Referring to, people such as Miller, BIOESSAYS (1989) 11 (4): 91.
Transfection can be finished by electroporation.Referring to, TROTTER and WOOD, 39 METHODS INMOLECULAR BIOLOGY (1995); Mann and King, J.GEN.VIROL. (1989) 70:3501.Perhaps, can use liposome with recombinant expression vector and baculovirus transfection insect cell.Referring to, for example, people such as Liebman, BIOTECHNIQUES (1999) 26 (1): 36; People such as Graves, BIOCHEMISTRY (1998) 37:6050; People such as Nomura, J.BIOL.CHEM. (1998) 273 (22): 13570; People such as Schmidt, PROTEINEXPRESSION AND PURIFICATION (1998) 12:323; People such as Siffert, NATURE GENETICS (1998) 18:45; People such as TILKTNS, CELL BIOLOGY:A LABORATORY HANDBOOK 145-154 (1998); People such as Cai, PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263; People such as Dolphin, NATURE GENETICS (1997) 17:491; People such as Kost, GENE (1997) 190:139; People such as Jakobsson, J.BIOL.CHEM. (1996) 271:22203; People such as Rowles, J.BIOL.CHEM. (1996) 271 (37): 22376; People such as Reverey, J.B IOL.CHEM. (1996) 271 (39): 23607-10; People such as Stanley, J.B IOL.CHEM. (1995) 270:4121; People such as Sisk, J.VIROL. (1994) 68 (2): 766; With people such as Peng, BIOTECHNIQUES (1993) 14 (2): 274.The liposome that can buy comprise (for example) Cellfectin  and Lipofectin  (Invitrogen, Corp., Carlsbad, CA).In addition, can use calcium phosphate transfection.Referring to, TROTTER and WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990) 18 (19): 5667; With Mann and King, J.GEN.VIROL. (1989) 70:3501.
Rhabdovirus expression vector contains bacilliform virus promoter usually.Bacilliform virus promoter is anyly can and cause the dna sequence dna that encoding sequence (for example, structure gene) downstream (3 ') is transcribed into mRNA in conjunction with shaft-like viral rna polymerase.Promotor will have and be usually located at the transcription initiation zone of approaching 5 of encoding sequence ' end most.Described transcription initiation zone generally includes RNA polymerase combining site and transcription initiation position.Bacilliform virus promoter also can have second territory that is called enhanser, if exist, it is usually away from structure gene so.In addition, express can be adjustment type or for composing type.
The structure gene of fully transcribing in the infection round-robin later stage provides useful especially promoter sequence.Example comprises gene (people such as FRIESEN, the The Regulation of Baculovirus GeneExpression in THE MOLECULAR BIOLOGY OF BACULOVIRUSES (1986) that is derived from the viral polyhedrin protein of coding; EP 0 127 839 and 0 155 476) and the sequence of coding p10 proteinic gene people such as (, J.GEN.VIROL. (1988) 69:765) Vlak.
The rhabdovirus expression vector that newly forms is packaged in the infectious recombinant baculovirus, and the plaque that can come purifying to grow by the known technology of those skilled in the art subsequently.Referring to, people such as Miller, BIOESSAYS (1989) 11 (4): 91; SUMMERS and SMITH, TEXAS AGRICULTURAL EXPERIMENTSTATION BULLETIN NO.1555 (1987).
Developed the recombination rhabdovirus expression vector that is used for infecting some insect cells.For example, developed the recombinant baculovirus that is particularly useful for Aedes aegypti (ATCC CCL-125 number), silkworm (ATCC CRL-8910 number), drosophila melanogaster (No. the 1963rd, ATCC), fall army worm and cabbage looper.Referring to, Wright, N ATURE(1986) 321:718; People such as Carbonell, J.VIROL. (1985) 56:153; People such as Smith, MOL.CELL.BIOL. (1983) 3:2156.Generally referring to, people such as Fraser, I NVITRO CELL.DEV.BIOL. (1989) 25:225.More particularly, the clone that is used for the rhabdovirus expression vector system generally includes (but being not limited to) Sf9 (fall army worm) (ATCC CRL-1711 number), Sf21 (fall army worm) (Invitrogen Corp., catalogue 11497-013 number (Carlsbad, CA)), Tri-368 (cabbage looper) and High-Five TMBTI-TN-5B1-4 (cabbage looper).
Be used for to buy, and cell culture technology generally is known concerning the those skilled in the art at the cell and the substratum of direct expression of baculovirus/expression and amalgamation and expression heterologous polypeptide.
Intestinal bacteria, pseudomonas kind and other prokaryotic organismThe bacterial expression technology is known to the those skilled in the art.The various carriers that are used for host bacterium are obtainable.Carrier can be single copy or low multiple copied or height multi-copy vector.Carrier can be used for the clone and/or express.In view of about the commercial availability of the lot of documents of carrier, many carriers and in view of the handbook of describing carrier and its restriction map and feature, do not need extensive discussions at this.As is well known, carrier is usually directed to the marker of selective usefulness, and described marker can provide cytotoxic agent resistance, former nutrition or immunity.Usually there are a plurality of markers that different characteristics is provided.
The bacterium promotor is anyly can and cause the dna sequence dna that encoding sequence (for example, structure gene) downstream (3 ') is transcribed into mRNA in conjunction with the bacteria RNA polysaccharase.Promotor will have and be usually located at the transcription initiation zone of approaching 5 of encoding sequence ' end most.Described transcription initiation zone generally includes RNA polymerase combining site and transcription initiation position.The bacterium promotor also can have second territory that is called operon, and it can be overlapping with the synthetic contiguous RNA polymerase combining site that begins to locate of RNA.Because gene repression albumen can in conjunction with operon and and then suppress transcribing of specific gene, so operon is allowed transcribing of negative regulation (derivable).Constitutive expression can take place when the negative regulatory element that does not exist such as operon.In addition, just regulating and control and can reach by the gene activation protein binding sequence, if described gene activation protein binding sequence exists, it approaches RNA polymerase binding sequence (5 ') usually most so.The proteic example of gene activation is catabolite activated protein (CAP), and it help to cause the lac operon at bacillus coli (intestinal bacteria, transcribing in E.coli) [people such as Raibaud, ANNU.R EV.G ENET. (1984) 18:173].Therefore expression through regulation and control can be positively charged or negative, and then strengthens or reduce and transcribe.
The sequence of coding metabolic pathway enzyme provides useful especially promoter sequence.Example comprises the promoter sequence that is derived from such as the carbohydrate metabolism enzyme of semi-lactosi, lactose (lac) [people such as Chang, NATURE (1977) 198:1056] and maltose.Other example comprises promoter sequence [people such as Goeddel, Nuc.ACIDS RES. (1980) 8:4057 that is derived from such as the biosynthetic enzyme of tryptophane (trp); People such as Yelverton, NUCL.ACIDS RES. (1981) 9:731; United States Patent (USP) the 4th, 738, No. 921; The open case of European patent the 036 No. 776 and the 121 No. 775, it is to be incorporated herein by reference].People such as beta-galactosidase enzymes (bla) promoter systems [Weissmann (1981) " The cloning of interferonand other mistakes. " In Interferon 3 (I.Gresser volume)], phage PL[Shimatake, NATURE (1981) 292:128] and T5[United States Patent (USP) the 4th, 689, No. 406, it is to be incorporated herein by reference] promoter systems also provides the promoter sequence of usefulness.The preferred method of the present invention utilization such as the strong promoter of T7 promotor is induced high-caliber GH (for example, hGH) polypeptide.The example of described carrier is known to the those skilled in the art and comprises from the pET29 of Novagen series and be described in pPOP carrier among the WO99/05297 that incorporates this paper by reference into.Described expression system produces high-caliber GH (for example, hGH) polypeptide, and can not jeopardize host cell viability or growth parameter(s) in the host.PET19 (Novagen) is a known another kind of carrier in this technology.
In addition, also play the bacterium promotor at the non-existent synthetic promoter of occurring in nature.For example, the transcription-activating sequence of bacterium promotor or phage promoter can engage with the operon sequence of another bacterium promotor or phage promoter, produces synthetic hybrid promoters [United States Patent (USP) the 4th, 551, No. 433, it is to be incorporated herein by reference].For example, the tac promotor is the hybridization trp-lac promotor of being made up of trp promotor and lac operon sequence that is subjected to lac repressor regulation and control [people such as Amann, GENE (1983) 25:167; People such as de Boer, PROC.N ATL.A CAD.SCI. (1983) 80:21].In addition, the bacterium promotor can comprise the naturally occurring promotor of the non-bacterial origin with the ability of transcribing in conjunction with bacteria RNA polysaccharase and initiation.The naturally occurring promotor of non-bacterial origin also can with compatible RNA polymerase coupling to produce the high level expression of some genes in prokaryotic organism.Phage t7 RNA polymerase/promoter systems is through the example of the promoter systems of coupling [people such as Studier, J.MOL.BIOL. (1986) 189:113; People such as Tabor, Proc Natl.Acad.Sci. (1985) 82:1074].In addition, hybrid promoters also can be made of (No. the 267 851, the open case of EP) phage promoter and intestinal bacteria operon zone.
Except that the function on subsequence, effectively ribosome binding site also can be used for the expression of foreign gene in prokaryotic organism.In intestinal bacteria, ribosome binding site is called as Xia Na-Doug promise (Shine-Dalgarno, SD) sequence and the length that comprises initiator codon (ATG) and be positioned upstream 3-11 Nucleotide place of initiator codon are sequence [people such as Shine, the N of 3-9 Nucleotide ATURE(1975) 254:34].Think that the SD sequence promotes mRNA to combine [people " Geneticsignals and nucleotide sequences in messenger RNA " such as Steitz with rrna by the base pairing between SD sequence and 3 of the intestinal bacteria 16S rRNA ' end, in Biological Regulation andDevelopment:Gene Expression (R.F.Goldberger compiles, 1979)].Express eukaryotic gene and prokaryotic gene [people such as Sambrook with weak ribosome binding site, " Expression of cloned genes inEscherichia coli ", Molecular Cloning:A Laboratory Manual, 1989].
Term " host bacterium " or " bacterial host cell " refer to the bacterium that can be used as or be used as the recipient of recombinant vectors or other transfer DNA.Described term comprises the filial generation of the primitive bacteria host cell of transfection.Should be appreciated that because sudden change accidental or that have a mind to, the filial generation of single parental cell is aspect morphology or can be identical aspect original parental generation complementary genome DNA or total DNA.With treat by (for example, hGH) filial generation of the quite similar parental cell of the parental generation that characterizes of the relevant nature of the existence of the nucleotide sequence of polypeptide is included in filial generation of this definition indication such as coding GH.
(for example, hGH) selection of the suitable host bacterium of polypeptide is known to the those skilled in the art to be used to express GH.When selecting the host bacterium that is used to express, appropriate host can comprise that demonstration especially has those hosts that good inclusion body forms ability, low proteolytic activity and whole robustness.Host bacterium generally can obtain from the various sources that include, but is not limited to following source: Bacterial Genetic Stock Center, Department ofBiophysics and Medical Physics, University of California (Berkeley, CA); With American TypeCulture Collection (" ATCC ") (Manassas, VA).The fermentation of industrial fermentation/medicine is general to be used and is derived from K bacterial strain (for example, bacterium W3110) or be derived from B bacterial strain (for example, bacterium BL21).Because the growth parameter(s) of these bacterial strains be very know with firm, so it is particularly useful.In addition, these bacterial strains are non-virulents, for this point of security and environment reason commercial very important.Other example of suitable escherichia coli host includes, but is not limited to the bacterial strain of BL21, DH10B or derivatives thereof.In another embodiment of method of the present invention, escherichia coli host is the negative bacterial strain of proteolytic enzyme, includes, but is not limited to OMP-and LON-.The host cell bacterial strain can be the pseudomonas kind, includes, but is not limited to Pseudomonas fluorescens, Pseudomonas aeruginosa and pseudomonas putida.The Pseudomonas fluorescens biotype 1 of known called after bacterial strain MB101 can be used for recombinant production and can be used for the therapeutic protein production technique.The example of pseudomonas expression system comprises the system as host strain (Midland, MI can obtain) that obtains from Dow Chemical Company on www.dow.com.The United States Patent (USP) that is incorporated herein by reference is described the pseudomonas bacterial strain for the 4th, 755, No. 465 and the 4th, 859, No. 600 as being used for GH (for example, the purposes of the host cell that hGH) produces.
In case the recombinant host cell bacterial strain is set up (promptly, expression constructs has been introduced in the host cell and to have a host cell of suitable expression constructs separated) after, just (for example, hGH) cultivate the recombinant host cell bacterial strain under the condition of polypeptide being suitable for producing GH.As be easy to being appreciated by one of skill in the art that, cultivate the method for recombinant host cell bacterial strain and will decide on the character of the expression constructs utilized and the characteristic of host cell.Usually use the known method of those skilled in the art to cultivate the recombinant host bacterial strain.Recombinant host cell is normally cultivated at the assimilable source that contains carbon, nitrogen and inorganic salt and (optionally) and is contained in the liquid nutrient medium of VITAMIN, amino acid, somatomedin and other those skilled in the art's known protein matter cultivation fill-in.The liquid nutrient medium that is used to cultivate host cell can optionally contain in order to microbiotic or anti-mycotic agent that prevents undesirable microorganism growth and/or the compound that includes, but is not limited to microbiotic (in order to select to contain the host cell of expression vector).
Recombinant host cell can be in batches or is cultivated with continuous form, and cell harvesting (is form in batches or continuously at the GH (for example, hGH) situation of polypeptide cell inner accumulated under) or the collection of culture supernatants simultaneously.Concerning producing in prokaryotic host cell, batch culture and cell harvesting are preferred.
(for example, hGH) polypeptide is purified after normally expressing in recombination system GH of the present invention.(for example, hGH) polypeptide can be by the purifying in addition from host cell or substratum of known the whole bag of tricks in this technology for GH.(for example, hGH) polypeptide may be (being the inclusion body form) indissoluble or insoluble to the GH that produces in bacterial host cell.In one embodiment of the invention, can be easily (for example at GH, hGH) carry out aminoacid replacement in the polypeptide, selecting described replacement is that known those methods increase the proteinic solvability that reorganization produces in method disclosed herein and this technology in order to utilize.Under the situation of insoluble protein, protein can be collected and further homogenizing succeeded by cell from the host cell lysate by centrifugal.Under the proteinic situation of insoluble, can add the compound that includes, but is not limited to polymine (PEI) and precipitate to induce partly soluble protein.Sedimentary protein can be collected by centrifugation then expediently.Can use the known the whole bag of tricks of those skilled in the art to make recombinant host cell broken or homogenize in cell, to discharge inclusion body.The technology of knowing that can use and include, but is not limited to enzymatic cytoclasis, supersound process, Du Ensi (dounce) homogenizes or high pressure release is broken is carried out the broken or process that homogenizes of host cell.In an embodiment of method of the present invention, use the high pressure release tech to make the e. coli host cell fragmentation to discharge GH (for example, the hGH) inclusion body of polypeptide.(for example, hGH) during the inclusion body of polypeptide, can advantageously make the multiple time of homogenizing be reduced to the shortest, and not have the loss that causes owing to factor handling GH such as solubilising, mechanical shearing or proteolysis so that the output of inclusion body reaches maximum.
Then can use in this technology any in known many suitable solubilizing agent to make insoluble or sedimentary GH (for example, hGH) polypeptide dissolving.Can make GH (for example, hGH) polypeptide dissolving with urea or Guanidinium hydrochloride.(for example, hGH) volume of polypeptide minimizes so that can use the batch weight of being convenient to manage to come large quantities of generations should to make the dissolved GH of institute.This factor can volume be may be very important during the large-scale commercial applications of the batch growth of thousands of liters is provided with at recombinant host.In addition, (for example, hGH) during polypeptide, during in particular for human medicinal use, should avoid to damage the harsh chemical product (if possible) of machine and container or protein itself when make GH with large-scale commercial applications setting.Shown that in the method for the invention relatively mild denaturing agent urea can be used to replace harsh denaturing agent Guanidinium hydrochloride and makes GH (for example, hGH) polypeptide inclusion body dissolving.The use meeting of urea significantly reduces (for example, hGH) stainless steel equipment that is utilized in the manufacturing of polypeptide and the purge process causes the risk of damage, makes GH (for example, hGH) polypeptide inclusion body dissolving simultaneously effectively at GH.
Under the proteinic situation of solubility hGH, hGH can be secreted in the periplasmic space or be secreted in the substratum.In addition, solubility hGH may reside in the tenuigenin of host cell.Before carrying out purification step, may need solubility hGH is concentrated.The known standard technique of those skilled in the art can be used for making the solubility hGH from (for example) cellular lysate or substratum to concentrate.In addition, the known standard technique of those skilled in the art can be used to make host cell broken and discharge solubility hGH from the tenuigenin or the periplasmic space of host cell.
When GH (for example, when hGH) polypeptide produces as fusion rotein, can remove fusion sequence.Removing of fusion sequence can be reached by enzymatic lysis or chemical cracking.The enzymatic of fusion sequence removes and can use the known method of those skilled in the art to reach.The selection that is used to remove the enzyme of fusion sequence will be determined by the characteristic that merges, and as will being easy to being appreciated by one of skill in the art that, reaction conditions will be determined by the selection of enzyme.Chemical cracking can use the known reagent that includes, but is not limited to cyanogen bromide, TEV proteolytic enzyme and other reagent of those skilled in the art to finish.(for example, hGH) polypeptide can be by the known method of those skilled in the art from purifying through the cracked fusion sequence through cracked GH.As will being easy to being appreciated by one of skill in the art that, described method will (for example, hGH) characteristic of polypeptide and character be determined by fusion sequence and GH.The method that is used for purifying can include, but is not limited to size exclusion chromatography,, hydrophobic interaction chromatography, ion exchange chromatography or dialysis or its any combination.
Also can with GH (for example, hGH) peptide purification to remove DNA from protein soln.DNA can also can remove by using the nucleic acid precipitation agent generation precipitation such as (but being not limited to) protamine sulfate by removing such as known any appropriate method in this technology of precipitation or ion exchange chromatography.(for example, hGH) polypeptide separates with sedimentary DNA can to use the well-known process that includes, but is not limited to centrifugal or filtering standard to make GH.Host's nucleic acid molecule remove that (for example, hGH) to wait to be used for to treat in the human setting be an important factor to polypeptide, and method of the present invention makes host cell DNA be reduced to pharmaceutically acceptable level at GH.
On a small scale the method for fermentation or large scale fermentation also can be used for protein expression, includes, but is not limited to fermentor tank, shakes bottle, fluidized bed bio reactor, hollow-fiber bioreactor, rolling bottle culture systems and stirring slot type bioreactor system.Each method in these methods can batch processes, charging-batch processes or continuous mode method are carried out.
Human GH polypeptide of the present invention generally can use the standard method in this technology to reclaim.For example, substratum or cellular lysate can be through centrifugal or filter to remove cell debris.Can with supernatant concentration or be diluted to the volume of wanting or filter thoroughly in the suitable damping fluid to regulate preparation for being further purified.(for example, hGH) being further purified of polypeptide comprises and makes GH (for example, hGH) the deamidated form of polypeptide variants is separated with complete form with clipped form GH of the present invention.
Can adopt any program in the following exemplary sequence to come purifying GH of the present invention (for example, hGH) polypeptide: affinity chromatography; Negatively charged ion or cation-exchange chromatography (use includes, but is not limited to DEAE SEPHAROSE); The silica chromatography; Reversed-phase HPLC; Gel-filtration (use includes, but is not limited to SEPHADEX G-75); The hydrophobic interaction chromatography; Size exclusion chromatography; Metal chelate chromatography; Ultrafiltration/diafiltration; Ethanol sedimentation; Ammonium sulfate precipitation; Chromatofocusing; Displacement chromatography; Electrophoretic procedures (including, but is not limited to the isoelectrofocusing of preparation property), difference solvability method (including, but is not limited to ammonium sulfate precipitation), SDS-PAGE or extraction method.
Protein of the present invention, include, but is not limited to comprise alpha-non-natural amino acid protein, comprise alpha-non-natural amino acid peptide, comprise alpha-non-natural amino acid proteinic antibody, comprise combination of proteins collocation thing of alpha-non-natural amino acid or the like, can come to be purified to partly or substantially homogeneous according to the known and employed standard program of those skilled in the art.Therefore, polypeptide of the present invention can reclaim and purifying by any method in the known many methods of those skilled in the art that include, but is not limited to ammonium sulfate or ethanol sedimentation, acid or alkali extraction process, column chromatography, affinity column chromatography method, negatively charged ion or cation-exchange chromatography, phosphocellulose chromatography method, hydrophobic interaction chromatography, hydroxyapatite chromatography method, Sugar receptors chromatography, gel electrophoresis etc.Can when making correctly folding mature protein, use the protein refolding step on demand.In the highly purified final purification step of needs, can adopt high performance liquid chromatography (HPLC), affinity chromatography or other suitable method.In one embodiment, the antibody of the anti-alpha-non-natural amino acid of the manufacturing protein or the peptide of alpha-non-natural amino acid (or comprise) as purified reagent, is included, but is not limited to be used to comprise the purified reagent based on the purifying of avidity of the protein of one or more alpha-non-natural amino acids or peptide.In case polypeptide by purifying partly or after being purified to homogeneous, is just optionally used it for multiple function on demand, include, but is not limited to as calibrating component, therapeutical agent, preventive, diagnostic reagent, scientific research reagent and/or the immunogen that produces as antibody.
Except that other mentioned reference of this paper, various purifying/protein folding method is known to the those skilled in the art, include, but is not limited to below with reference to those methods described in the document: R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology, the 182nd volume: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana, (1997) Bioseparation of Proteins, Academic Press, Inc.; People such as Bollag (1996) Protein Methods, the 2nd edition, Wiley-Liss, NY; Walker, (1996) The Protein Protocols HandbookHumana Press, NJ; Harris and Angal, (1990) Protein Purification Applications:A Practical ApproachIRL Press atOxford, Oxford, England; Harris and Angal, Protein Purification Methods:A Practical ApproachIRL Press at Oxford, Oxford, England; Scopes, (1993) Protein Purification: Principles and Practice, the 3rd edition, Springer Verlag, NY; Janson and Ryden, (1998) Protein Purification:Principles, High Resolution Methods and Applications, the 2nd edition,Wiley-VCH, NY; And Walker (1998), Protein Protocols on CD-ROMHumana Press, NJ; The reference of being quoted wherein.
Producing the protein with alpha-non-natural amino acid paid close attention to or an advantage of polypeptide in eukaryotic host cell or non-eukaryotic host cell is that protein or polypeptide will be folded into its native conformation usually.Yet, in certain embodiments of the present invention, those skilled in the art will realize that behind synthetic, expression and/or purifying, protein or peptide can have with related polypeptide the different conformation of the conformation of wanting.In one aspect of the invention, optionally make expressed protein denaturation and make its renaturation subsequently.This is to utilize that known method realizes in this technology, include, but is not limited to by chaperone (chaperonin) is added in the protein of being paid close attention to or polypeptide, by protein is dissolved in the chaotropic agent such as Guanidinium hydrochloride, realize by utilizing protein disulfide isomerase to wait.
In general, need to make expressed polypeptide sex change with reduction and then make the polypeptide refolding become preferred conformation sometimes.For example, guanidine, urea, DTT, DTE and/or chaperone can be added in the translation product of being paid close attention to.The method that makes protein reduction, sex change and renaturation to the those skilled in the art be known (referring to, people such as above-mentioned reference and Debinski, (1993) J.Biol.Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconiug.Chem., 4:581-585; With people such as Buchner, (1992) Anal.Biochem.,205:263-270).For example, people such as Debinski describes sex change and the reduction of inclusion body protein in guanidine-DTE.Protein can include, but is not limited to refolding in Sleep-promoting factor B and the arginic potential buffer solution of L-containing.Refolding reagent is flowed or move contacting, or vice versa with one or more polypeptide or other expression product in other mode.
Protokaryon produce GH (for example, hGH) under the situation of polypeptide, so the GH that produces (for example, hGH) polypeptide can be false folding and therefore lack biological activity or have the biological activity of reduction.Proteinic biological activity can be recovered by " refolding ".In general, the GH of false folding (for example, hGH) polypeptide is by (for example using (for example) one or more chaotropic agents, urea and/or guanidine) and the reductive agent that can make disulfide bond reduction is (for example, dithiothreitol (DTT), DTT or 2 mercapto ethanol, 2-ME) make polypeptide chain dissolving (wherein GH (for example, hGH) polypeptide also is insoluble), separate folding and reduction and refolding.Then add oxygenant (for example, oxygen, Gelucystine or cystamine) under the chaotropic agent of intermediate concentration, it makes disulfide linkage form again.(for example, hGH) polypeptide can use known standard method in this technology and refolding to GH, such as in the United States Patent (USP) of incorporating this paper by reference into the 4th, 511, No. 502, the 4th, 511, No. 503 and the 4th, 512, those methods described in No. 922.(for example, hGH) polypeptide also can fold and formation heterodimer or heteropolymer with other protein GH altogether.
Refolding or altogether folding after, can (for example, hGH) polypeptide be further purified to GH.GH (for example, hGH) purifying can use the known various technology of those skilled in the art to realize that described technology comprises hydrophobic interaction chromatography, size exclusion chromatography,, ion exchange chromatography, reversed-phased high performace liquid chromatographic, affinity chromatography etc. or its any combination.Extra purifying also can comprise makes the dry or sedimentary step of purified protein.
Behind purifying, GH (for example, hGH) can be switched in the different damping fluids and/or is concentrated by any method in the known several different methods in this technology that includes, but is not limited to filter and dialysis.The GH that provides with the protein form of single purifying (for example, hGH) can stand to assemble and precipitation.
Purified GH (for example, hGH) can be at least 90% pure (as measured) or at least 95% pure by reversed-phased high performace liquid chromatographic RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE, or at least 98% is pure, or at least 99% or 99% is above pure.No matter GH (for example, the definite numerical value of purity hGH) is what, to as pharmaceutical prod or be used for such as with water-soluble polymers (such as, bonded PEG) is processing further, GH (hGH) is enough pure for example.
(for example, hGH) molecule can not exist other activeconstituents or protein to be used as therapeutical agent when (being different from vehicle, supporting agent and stablizer, serum albumin etc.) to some GH, or it can be compound with another kind of protein or polymkeric substance.
General purification processAny step in the various separating steps can be at (for example comprising GH, hGH) cellular lysate of the host cell of polypeptide, extract, substratum, inclusion body, periplasmic space, the tenuigenin of host cell or other material are carried out, or (for example at any GH that obtains by any separating step, hGH) polypeptide mixture is carried out, described separating step includes, but is not limited to affinity chromatography, ion-exchange chromatography, the hydrophobic interaction chromatography, gel filtration chromatography, high performance liquid chromatography (" HPLC "), reversed-phase HPLC (" RP-HPLC "), Expanded Bed Adsorption or its any combination and/or repetition, and can any suitable order carry out.
Employed equipment must want material to buy with other in carrying out the techniques described herein.Pump, fraction collector, monitor, register and holonomic system can be from (for example) Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway NJ) obtains.The chromatographic material that includes, but is not limited to exchange group material, substratum and damping fluid also can obtain from described company.
Such as the balance in the described herein column chromatography of washing and wash-out and other step, can use such as the specific equipment of pump and finish more quickly.The pump that can buy includes, but is not limited to HILOAD Pump P-50, peristaltic pump P-1, pump P-901 and pump P-903 (Amersham Biosciences, Piscataway, NJ).
The example of fraction collector comprise RediFrac fraction collector, FRAC-100 and FRAC-200 fraction collector and SUPERFRAC  fraction collector (Amersham Biosciences, Piscataway, NJ).Mixing tank also is obtainable to form pH value and linear concentration gradient.The mixing tank that can buy comprise mixing tank on gradient mixer GM-1 and the line (In-Line Mixer) (Amersham Biosciences, Piscataway, NJ).
The chromatography process can use any monitor of buying to monitor.Described monitor can be used to collect the information as UV, pH value and specific conductivity.The example of detector comprise monitor UV-1, UVICORD  S II, monitor UV-MII, monitor UV-900, monitor UPC-900, monitor pH/C-900 and monitored conductivity device (AmershamBiosciences, Piscataway, NJ).In fact, comprise that (Piscataway, the holonomic system of various AKTA  system NJ) can be buied from Amersham Biosciences.
In one embodiment of the invention, for example, GH (for example, hGH) the purified GH that polypeptide can be by at first making gained (for example, hGH) polypeptide sex change in urea then is diluted to and contains reductive agent (such as in the suitable TRIS damping fluid of, pH value DTT) and reduction and sex change.In another embodiment, (for example, hGH) polypeptide is about 2M sex change in the urea between about 9M in concentration range, then is diluted to the pH value in about 5.0 TRIS damping fluids in about 8.0 scopes to make GH.Can cultivate the refolding mixture of described embodiment then.In one embodiment, the refolding mixture is at room temperature cultivated 4 hours to 24 hours.Then can be to (for example, hGH) polypeptide mixture carries out further isolated or purified through reduction and the GH of sex change.
As described herein, can (for example, hGH) the pH value of polypeptide mixture be regulated, and carries out any separating step subsequently then to a GH.In addition, (for example, hGH) polypeptide mixture or its any mixture subsequently concentrate can to use in this technology known technology to make a GH.In addition, (for example, hGH) elution buffer of polypeptide mixture or its any mixture subsequently changes the damping fluid that is suitable for next separating step into can to use the known technology of those skilled in the art will comprise a GH.
Ion-exchange chromatographyIn one embodiment and as optional, an extra step, can (for example, hGH) polypeptide mixture be carried out ion-exchange chromatography at a GH.Generally referring to ION EXCHANGECHROMATOGRAPHY:PRINCIPLES AND METHODS (catalogue 18-1114-21 number, AmershamBiosciences (Piscataway, NJ)).The ion exchange column that can buy comprises HITRAP , HIPREP And HILOAD Post (Amersham Biosciences, Piscataway, NJ).Described post utilizes strong anion exchanger, such as, Q SEPHAROSE Fast Flow, Q SEPHAROSE High Performance and Q SEPHAROSE  XL; Strong cation exchanger, such as, SP SEPHAROSE High Performance, SP SEPHAROSE FastFlow and SP SEPHAROSE XL; Weak anion exchanger, such as, DEAE SEPHAROSE Fast Flow; And weak cation exchanger, such as, CM SEPHAROSE Fast Flow (Amersham Biosciences, Piscataway, NJ).Can be when any stage of purge process (for example, hGH) polypeptide is carried out negatively charged ion or cation exchange column chromatography method, to separate substantially the GH of purifying (for example, hGH) polypeptide at GH.The cation-exchange chromatography step can use any suitable cationic exchange matrix to carry out.That available cationic exchange matrix includes, but is not limited to is fibrous, porous, cationic exchange substrate material atresia, particulate, pearl or crosslinked.Described cationic exchange substrate material includes, but is not limited to the matrix material of Mierocrystalline cellulose, agarose, dextran, polyacrylic ester, polyethylene, polystyrene, silica, polyethers or any previous materials.
Cationic exchange matrix can be any suitable cationite that comprises strong cation exchanger and weak cation exchanger.Strong cation exchanger can keep ionization state and thereby can be in wide pH value scope in conjunction with GH (for example, hGH) in wide pH value scope.Yet weak cation exchanger can lose ionization with the variation of pH value.For example, when pH is reduced to about pH4 or pH5 when following, weak cation exchanger may lose electric charge.Suitable strong cation exchanger includes, but is not limited to the charged functional groups such as sulfopropyl (SP), methylmesylate (S) or sulfoethyl (SE).Cationic exchange matrix can be preferably to have about 2.5 to about 6.0 GH (for example, hGH) in conjunction with the strong cation exchanger of pH value scope.Perhaps, strong cation exchanger can have about pH2.5 to the GH of about pH5.5 (for example, hGH) in conjunction with pH value scope.Cationic exchange matrix can be to have about 3.0 GH (for example, hGH) in conjunction with the strong cation exchanger of pH value.Perhaps, cationic exchange matrix can be preferably to have about 6.0 to about 8.0 GH (for example, hGH) in conjunction with the strong cation exchanger of pH value scope.Cationic exchange matrix can be preferably to have about 8.0 to about 12.5 GH (for example, hGH) in conjunction with the strong cation exchanger of pH value scope.Perhaps, strong cation exchanger can have about pH8.0 to the GH of about pH12.0 (for example, hGH) in conjunction with pH value scope.
(for example, hGH) before, can (for example) use the weak acid (for example, the pH value of 4 column volumes is 3 20mM acetate) of the dilution of some column volumes to make cationic exchange matrix balance at loading GH.After the balance, can add GH (for example, hGH), and can wash-out substantially the GH of purifying (for example, hGH) use again before such as the weakly acid soln of weak acetate or phosphoric acid solution several times once arrived in the pillar washing.For example, can use the pH value of about 2-4 column volume is that 3 20mM acetate washs pillar.The pH value that also can use 2-4 column volume of usefulness (for example) is that 5.5 0.05M sodium acetate or pH value are 5.5 and extra washing 0.1M sodium-chlor blended 0.05M sodium acetate.Perhaps, utilize known method in this technology, can use the weak base of the dilution of some column volumes to make cationic exchange matrix balance.
Perhaps, the GH of purifying (for example, hGH) can contact wash-out so that GH (for example, hGH) is cemented out from matrix with the damping fluid with enough low pH value or ionic strength by making cationite matrix substantially.The pH value of elution buffer can be about 2.5 to pH values in the pH value and be about in 6.0 the scope and change.More particularly, the pH value of elution buffer can be about that 2.5 to pH values are about 5.5 in the pH value, the pH value is about 2.5 to pH values and is about in 5.0 the scope and changes.Elution buffer can have about 3.0 pH value.In addition, the amount of elution buffer can significantly change and will be generally in about 2 column volumes arrive the scope of about 10 column volumes.
At GH (for example, hGH) after polypeptide is adsorbed in cationite matrix, the hGH polypeptide of purifying can contact wash-out so that (for example, hGH) polypeptide cements out from matrix with GH by making matrix and the damping fluid with sufficiently high pH value or ionic strength substantially.(for example, hGH) suitable buffer of the high pH value wash-out of polypeptide includes, but is not limited to concentration at the Citrate trianion, phosphoric acid salt, formate, acetate, HEPES and the MES damping fluid that change in the scope at least about 100mM at least about 5mM can be used for the GH of purifying substantially as used herein.
Reverse-phase chromatographyCan carry out RP-HPLC with protein purification according to the known suitable scheme of those skilled in the art.Referring to, for example, people such as Pearson, ANAL BIOCHEM. (1982) 124:217-230 (1982); People such as Rivier, J.CHROM. (1983) 268:112-119; People such as Kunitani, J.CHROM. (1986) 359:391-402.Can (for example, hGH) polypeptide be carried out RP-HPLC to separate substantially the GH of purifying (for example, hGH) polypeptide at GH.With regard to this point, can use to have multiple length and (include, but is not limited at least about C 3Arrive at least about C 30, at least about C 3Arrive at least about C 20, or at least about C 3Arrive at least about C 18) the alkyl functional base through silica deutero-resin.Perhaps, can use polymer resin.For example, can use TosoHaas AmberchromeCG1000sd resin as styrenic polymer resins.Also can use cyano group or polymer resin with multiple alkyl chain length.In addition, can be with the RP-HPLC post being washed such as the alcoholic acid solvent.Source RP post is another example of RP-HPLC post.
Contain ion-pairing agent and organic modifiers (such as, methyl alcohol, Virahol, tetrahydrofuran (THF), acetonitrile or ethanol) suitable elution buffer can be used to that (for example, hGH) polypeptide elutes from the RP-HPLC post with GH.The most frequently used ion-pairing agent includes, but is not limited to acetate, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, hyptafluorobutyric acid, triethylamine, tetramethylammonium, tetrabutylammonium and three second ammonium acetates.Can use one or more gradients or isocratic condition to carry out wash-out, wherein gradient condition shortens and peak width is reduced through preferred so that disengaging time.Another kind method relates to uses two kinds of gradients with different solvents concentration range.The example that can be used for suitable elution buffer herein can include, but is not limited to ammonium acetate and acetonitrile solution.
Hydrophobic interaction chromatography purification technologyCan (for example, hGH) polypeptide be carried out hydrophobic interaction chromatography (HIC) at GH.Generally referring to, HYDROPHOBIC INTERACTIONCHROMATOGRAPHY HANDBOOK:PRINCIPLES AND M ETHODS(catalogue 18-1020-90 number), (Piscataway, NJ), it is to be incorporated herein by reference to Amersham Biosciences.Appropriate H IC matrix can include, but is not limited to the matrix through the alkyl or aryl replacement, such as, through the matrix that butyl, hexyl, octyl group or phenyl replace, described matrix comprises agarose, Sepharose, sepharose, Mierocrystalline cellulose, silica, dextran, polystyrene, poly-(methacrylic ester) matrix; And hybrid resin, poly-(methacrylic ester) matrix that includes, but is not limited to the polyvinylamine resin or replace through butyl or phenyl.The commercially available source that is used for the hydrophobic interaction column chromatography includes, but is not limited to HITRAP , HIPREP And HILOAD Post (Amersham Biosciences, Piscataway, NJ).Briefly, before loading, can use the known standard buffer solution of those skilled in the art (such as, acetate/sodium chloride solution or contain the HEPES of ammonium sulfate) to make the HIC column equilibration.Ammonium sulfate can be as the damping fluid that loads the HIC post.(for example, hGH) behind the polypeptide, then can use standard buffer solution and condition that pillar is washed to remove unwanted material but make GH (for example, hGH) polypeptide remains on the HIC post loading GH.Can come wash-out GH (for example, hGH) polypeptide to the standard buffer solution of about 10 column volumes (especially such as the HEPES damping fluid that contains the low ammonium sulfate of EDTA and concentration ratio level pad, or acetate/sodium-chlor damping fluid) with about 3 column volumes.Use the linear salt gradient that successively decreases of (for example) potassiumphosphate gradient also to can be used to wash-out GH (for example, hGH) molecule.Can (for example) elutriant be concentrated then by filteration such as saturating filter or ultrafiltration.Can utilize filter to remove to be used for wash-out GH (for example, the hGH) salt of polypeptide.
Other purification technique can be at a GH (for example, hGH) polypeptide mixture or its any mixture are subsequently carried out and are used (for example) gel-filtration (GEL FILTRATION:PRINCIPLES AND METHODS (catalogue 18-1022-18 number, Amersham Biosciences, Piscataway, NJ), it is to be incorporated herein by reference), (suitable matrix includes, but is not limited to HA-Ultrogel to the hydroxyapatite chromatography method, High Resolution (Calbiochem), CHT pottery hydroxylapatite (BioRad), Bio-Gel HTP hydroxylapatite (BioRad)), HPLC, Expanded Bed Adsorption, ultrafiltration, saturating filter, another separating step of freeze-drying etc. to be removing any excessive salt and with the damping fluid before the suitable damping fluid displacement, to be used for next separating step or even to be used for the allotment of final medicament production.
Can use the monitoring when each step as herein described of the known technology of those skilled in the art to comprise substantially the GH of purifying (for example, the hGH) GH of polypeptide (for example, the hGH) productive rate of polypeptide.Described technology also can be used for assessing GH (for example, the hGH) productive rate of polypeptide of the purifying substantially behind the final separating step.For example, can use some anti-phase high pressure liquid chromatography with various alkyl chain length (such as, cyano group RP-HPLC, C 18RP-HPLC) any post in the post and cationic exchange HPLC and gel-filtration HPLC monitor GH (for example, the hGH) productive rate of polypeptide.
In certain embodiments of the invention, GH behind each purification step (for example, hGH) productive rate can be in the initial substance of each purification step GH (for example, hGH) at least about 30%, at least about 35%, 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%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, 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%, at least about 98%, at least about 99%, at least about 99.9% or at least about 99.99%.
Purity can be used such as the standard technique of SDS-PAGE and measure or by using west ink dot (Westernblot) and ELISA calibrating measurement GH (for example, hGH) polypeptide and measuring.For example, can produce anti-the recovery with cationic exchange and separate the proteinic polyclonal antibody that obtains from the negative control yeast fermentation.Antibody also can be used to survey the existence of polluting host cell proteins matter.
RP-HPLC material Vydac C4 (Vydac) is made up of silica gel particle, and its surface has the C4-alkyl chain.(for example, hGH) polypeptide separates the difference of the intensity that is based on hydrophobic interaction with protein impurity with GH.The acetonitrile gradient of use in rare trifluoroacetic acid carried out wash-out.Preparation property HPLC is to use stainless steel column (be equipped with 2.8 and be raised to 3.2 liters Vydac C4 silica gel) to carry out.Hydroxylapatite Ultrogel elutriant is the acidifying by adding trifluoroacetic acid, and it is loaded on the Vydac C4 post.For washing and wash-out, use the acetonitrile gradient in rare trifluoroacetic acid.Collect flow point and neutralize with phosphate buffered saline buffer immediately.Will be in (for example, hGH) the polypeptide flow point merging of the GH in the IPC limit.
DEAE sepharose (Pharmacia) material is by covalently forming with sepharose surface of beads bonded diethyllaminoethyl (DEAE).(for example, hGH) ionic interaction that is combined into of polypeptide and DEAE group mediates GH.Acetonitrile and trifluoroacetic acid do not keep by pillar.After these materials are washed off, remove trace impurity by acetate buffer washing pillar with low pH value.Then with neutral phosphonic phthalate buffer washing pillar and the buffer solution elution GH that increases with ionic strength (for example, hGH) polypeptide.Flow (DEAESepharose fast flow) filling pillar fast with the DEAE sepharose.The adjustable column volume with guarantee GH (for example, hGH) the polypeptide loading capacity at 3-10mg GH (for example, hGH) in the scope of polypeptide/ml gel.Water and level pad (sodium phosphate/potassiumphosphate) washing pillar.Loading washs pillar through the HPLC elutriant flow point of merging and with level pad.Then, with lavation buffer solution (sodium acetate buffer) washing pillar, then wash with level pad.Subsequently, with elution buffer (sodium-chlor, sodium phosphate/potassiumphosphate) from pillar wash-out GH (for example, hGH) polypeptide and collect with single flow point according to main elution profile.The elutriant of DEAE sepharose post is adjusted to the specific conductivity of regulation.Be aseptically filled in the teflon bottle (Teflon bottle) medicine of gained and storage under-70 ℃.
Other method that can adopt includes, but is not limited in order to remove endotoxic step.Intracellular toxin is the lipopolysaccharides (LPS) that is positioned at such as on the adventitia of the Gram-negative host cell of colon bacillus.The purification technique of known to the those skilled in the art being and the combination that include, but is not limited to use silica upholder, glass powder or hydroxylapatite, reverse-phase chromatography, affinity chromatography, size exclusion chromatography,, anion-exchange chromatography, hydrophobic interaction chromatography, these methods of the method that reduces level of endotoxin etc.May need to revise or need other method removing from the polypeptide of being paid close attention to such as the proteinic pollutent of migration altogether.The method that is used to measure level of endotoxin is known to the those skilled in the art and includes, but is not limited to king crab amebocyte lysate (LimulusAmebocyte Lysate) and (LAL) examine and determine.
Several different methods and program can be used to assess and (for example comprise one or more non-naturally encoded amino acid whose GH, hGH) proteinic productive rate and purity, described method and program comprise that (but being not limited to) Bradford (Bradford) calibrating, SDS-PAGE, silver dye SDS-PAGE, Xylene Brilliant Cyanine G (coomassie) dyeing SDS-PAGE, mass spectroscopy (including but not limited to MALDI-TOF) and known other method that is used for profiling protein matter of those skilled in the art.
Other method includes, but is not limited to: the SDS-PAGE that is used in combination with the protein staining method, immune ink dot method (immunoblotting), substance assistant laser desorpted/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry analytical method, isoelectrofocusing, analytical anionresin, chromatofocusing and circular dichroism spectrum.
VIII. the expression in the alternative system
Adopted some strategies with at the non-recombinant hosts cell, in the host cell that sudden change is handled or in cell free system, non-natural amino acid is introduced in the protein.These systems also are applicable to and make GH of the present invention (for example, hGH) polypeptide.The amino acid whose derivatize such as Lys, Cys and Tyr with reactive side chain can cause Methionin to be converted into N 2-ethanoyl-Methionin.Chemosynthesis also provides a kind of direct method of incorporating alpha-non-natural amino acid into.Along with the enzymatic of peptide fragment connects the newly-developed that is connected with native chemical, might make bigger protein.Referring to, for example, P.E.Dawson and S.B.H.Kent, Annu.Rev.Biochem, 69:923 (2000).The chemical peptide connects and is connected with native chemical is to be described in United States Patent (USP) the 6th, 184, among No. 344, No. the 2004/0138412nd, U.S. Patent Publication case, No. the 2003/0208046th, U.S. Patent Publication case, WO 02/098902 and the WO 03/042235, described reference is to be incorporated herein by reference.To join the general in vitro biosynthetic means that can keep in the biosynthetic in vitro extract of protein through the inhibition tRNA of desired alpha-non-natural amino acid chemical acidylate has been used to the alpha-non-natural amino acid site specific ground that surpasses 100 is incorporated in the range protein with virtually any size almost.Referring to, for example, 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, A general method forsite-specific incorporation of unnatural amino acids into proteins, Science244:182-188 (1989); And J.D.Bain, C.G. Glabe, T.A.Dix, A.R.Chamberlin, E.S.Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into a polypeptide, J.Am.Chem.Soc.111:8013-8014 (1989).The functional group with wide region introduces in the protein to be used to study protein stability, protein folding, enzyme mechanism and signal transduction.
Develop a kind of scrambling (promiscuity) of in vivo method that selection pressure is incorporated into that be called to utilize the wild-type synthetic enzyme.Referring to, for example, N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L. Moroder and R.Huber, FASEB J., 13:41 (1999).The cut auxotrophic strain in associated metabolic path of wherein supplying with the specific natural amino acid of cell is grown in the minimum medium of the natural amino acid that contains limited concentration, and transcribing of target gene is suppressed simultaneously.When stationary growth phase began, natural amino acid was depleted and replaced by the alpha-non-natural amino acid analogue.Induce the expression of recombinant protein to cause containing the proteinic accumulation of non-natural analogue.For example, use this strategy, incorporated into adjacent fluorophenylalanine, a fluorophenylalanine and P-fluoropnenylalanine in the protein and in UV spectrum, presented two characteristics being differentiated easily and takeed on the shape projections, referring to, for example, C.Minks, R.Huber, L.Moroder and N.Budisa Anal.Biochem., 284:29 (2000); The fluoroform methyllanthionine has been used for replacing methionine(Met) in the phage T4 N,O-Diacetylmuramidase so that pass through 19F NMR studies the interaction of itself and oligochitosan ligand, referring to, for example, H.Duewel, E.Daub, V.Robinson and J.F.Honek, Biochemistry, 36:3404 (1997); And the trifluoro leucine has been incorporated into the replacement leucine, thereby caused the thermostability of leucine zipper protein and chemical stability to increase.Referring to, for example, 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, selenomethionine and telluro methionine(Met) are incorporated in the various recombinant proteins in the X ray crystal analysis, to promote dissolving mutually.Referring to, for example, 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, L.Lebioda 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 methionine(Met) analogue that will have alkene or alkynes functional group is incorporated into effectively, thereby allows by chemical process protein to be done to modify in addition.Referring to, for example, J.C.van Hest and D.A.Tirrell, FEBS Lett., 428:68 (1998); J.C.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); United States Patent (USP) the 6th, 586, No. 207; U.S. Patent Publication case 2002/0042097, it is to be incorporated herein by reference.
The success of this method depends on that by the identification of aminoacyl-tRNA synthetase to the alpha-non-natural amino acid analogue generally speaking, described aminoacyl-tRNA synthetase needs highly selective to guarantee the fidelity of reproduction of protein translation.A kind of mode of expanding the scope of this method is to relax the substrate specificity of aminoacyl-tRNA synthetase, and it is achieved under condition of limited.For example, in bacillus coli phenylalanyl-tRNA synthetic enzyme (PheRS), replace Ala with Gly 294Can increase the size of substrate binding pocket, and cause tRNAPhe by fenclonine (p-Cl-Phe) acidylate.Referring to, M.Ibba, P.Kast and H.Hennecke, Biochemistry, 33:7107 (1994).Bacillus coli bacterial strain with this mutant PheRS allow incorporate fenclonine into or to bromophenyl alanine with the substituted benzene L-Ala.Referring to, for example, M.Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); With N.Sharma, R.Furter, P.Kast and D.A.Tirrell, FEBS Lett., 467:37 (2000).Similarly, the point mutation Phe130Ser that shows the amino acid combining site of close bacillus coli tyrosyl-tRNA synthetic enzyme allows that azatyrosine is more effectively incorporated into than tyrosine.Referring to, F. Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soll and S.Nishimura, J.Biol.Chem., 275:40324 (2000).
The another kind of strategy of in vivo alpha-non-natural amino acid being incorporated in the protein is to modify the synthetic enzyme with check and correction mechanism.These synthetic enzyme can not be distinguished amino acid similar to the homology natural amino acid on the structure and therefore can not activate described amino acid.This mistake is corrected at independent position, and it makes from the amino acid of the mischarging of tRNA and sloughs acyl group to keep the fidelity of reproduction of protein translation.If the check and correction loss of activity of synthetic enzyme can be avoided editting function and is merged in through wrong activatory analog so.Used valyl-tRNA synthetic enzyme (ValRS) to prove this method recently.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 use Cys, Thr or aminobutyric acid salt (Abu) with the amino acidylate of tRNAVal mistake; These non-homogeneous amino acid are hydrolyzed by edit field subsequently.Behind bacillus coli karyomit(e) generation random mutation, the editor position that is chosen in ValRS has the sudden change bacillus coli bacterial strain of sudden change.This editor's defective type ValRS loads tRNAVal with Cys improperly.Because Abu spatially be similar to Cys (Cys-the SH group in Abu-CH 3Replace), so when this sudden change bacillus coli bacterial strain was grown in the presence of Abu, sudden change ValRS also incorporated Abu in the protein into.Mass spectroscopy shows that in natural protein, about 24% Xie Ansuan is replaced by Abu in each Xie Ansuan position.
Solid phase synthesis and semisynthesis also allow to be used to contain a large amount of proteinic synthetic of new amino acid.For example, referring to following discloses case and the reference wherein quoted, as follows: 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 on polypeptides.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, Acc 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 means? Protein Eng., 1 (3): 151-157 (1987); And Jackson, D.Y., Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J.A.A Designed Peptide Ligase forTotal Synthesis of Ribonuclease A with Unnatural Catalytic Residues, Science, 266 (5183): 243 (1994).
Chemically modified has been used in vitro will comprising in the various non-natural side chains introducing protein of cofactor, spin labeling and oligonucleotide.Referring to, for example, Corey, D.R., Schultz, P.G. Generation of a hybridsequence-specific single-stranded deoxyribonuclease, Science, 238 (4832): 1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E.The chemical modification of enzymaticspecificity, Annu Rev Biochem, 54:565-595 (1985); Kaiser, E.T., Lawrence, D.S.Chemicalmutation of enyzme active sites, Science, 226 (4674): 505-511 (1984); Neet, K.E., Nanci A, Koshland, D.E.Properties of thiol-subtilisin, J Biol.Chem, 243 (24): 6392-6401 (1968); Polgar, L. et M.L.Bender.A new enzyme containing a synthetically formed active site.Thiol-subtilisin. J.Am Chem Soc, 88:3153-3154 (1966); And Pollack, S.J., Nakayama, G.Schultz, P.G. Introduction of nucleophiles and spectroscopic probes into antibody combiningsites, Science, 242 (4881): 1038-1040 (1988).
Perhaps, will adopt biosynthetic means to be used for some biophysics probes being incorporated in institute's synthetic protein in vitro through the aminoacyl-tRNA of chemically modified.Referring to following discloses case and the reference wherein quoted: 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 sequenceof nascent preprolactin of the 54-kilodalton polypeptide of the signal recognition particle Proc.Natl.Acad.Sci, 83 (22): 8604-8608 (1986).
Showed in the past, and can use in the protein synthesis reaction that contains the gene programization of wanting the amber nonsense mutation to some extent by joining, and in vitro alpha-non-natural amino acid site specific ground be incorporated in the protein through the inhibition tRNA of the amino acidylate of chemical.Utilize these methods, the those skilled in the art can use the specific amino acids auxotrophic strain to replace many 20 common seed amino acids (for example, with fluorophenylalanine substituted benzene L-Ala) with structure homologue in close relations.Referring to, for example, Noren, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G. Ageneral methodfor site-specific incorporation of unnatural amino acids into proteins, Science, 244:182-188 (1989): people such as M.W.Nowak, 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 aminoacid into a polypeptide, J.Am Chem Soc, 111:8013-8014 (1989); People such as N.Budisa, FASEB J.13:41-51 (1999); Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G.Biosynthetic method for introducing unnatural amino acids site-specifically into proteins. Methods in Enz., the 202nd volume, 301-336 (1992); And Mendel, D., Cornish, V.W.﹠amp; Schultz, P.G.Site-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys.Biomol Struct.24,435-62 (1995).
For example, the inhibition tRNA of preparation identification terminator codon UAG and with alpha-non-natural amino acid with the amino acidylate of its chemical.Terminator codon TAG is introduced at the position of being paid close attention to that the rite-directed mutagenesis of routine can be used in protein gene.Referring to, for example, Sayers, J.R., Schmidt, W.Eckstein, F.5 '-3 ' Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, Nucleic Acids Res, 16 (3): 791-802 (1988).In vitro transcribe/when making up in the translation system, alpha-non-natural amino acid is incorporated into when making, obtain containing that amino acid whose protein in specified location in response to the UAG codon through the inhibition tRNA and the mutator gene of acidylate.Use [ 3H]-experiment of Phe and incorporating desired amino acid into by UAG codon appointed positions place with only experiment showed, of alpha-hydroxy acid, and this seed amino acid is not incorporated at any other position in protein.Referring to, for example, people such as Noren, the same; People such as Kobayashi, (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 make the amino acidylate of tRNA with desired amino acid by any method or the technology that includes, but is not limited to the amino acidylate of chemical amino acidylate or enzymatic.
Amino acidylate can be finished by aminoacyl tRNA synthetase or by other enzymatic molecule that includes, but is not limited to ribozyme.Term " ribozyme " can exchange with " catalytic RNA ".Cech and colleague (Cech, 1987, Science, 236:1532-1539; People such as McCorkle, 1987, Concepts Biochem.64:221-226) proved the existence of the naturally occurring RNA (ribozyme) that can serve as catalyzer.Yet, can the Yeast Nucleic Acid substrate that be used for cracking and montage be worked although only shown these natural RNA catalyzer, the latest developments of the artificial evolution of ribozyme expand to various chemical reactions with the function (repertoire) of katalysis.Research differentiated can himself (2 ') 3 '-end on the RNA molecule (people such as Illangakekare of catalysis aminoacyl-RNA key; 1995 Science 267:643-647) and can with amino acid from a RNA molecular transfer to another RNA RNA molecule molecule (people such as Lohse; 1996, Nature 381:442-444).
The U.S. Patent Application Publication case that is incorporated herein by reference 2003/0228593 is described the method that makes up ribozyme and its makes tRNA that purposes in the amino acidylate takes place with natural coding and non-naturally encoded amino acid.The substrate immobilization form of enzymatic molecule that includes, but is not limited to the amino acidylate of made tRNA of ribozyme can make through effective affinity purification of the product of amino acidylate and can realize.The example of suitable substrate comprises agarose, sepharose and magnetic beads.The generation and the purposes of substrate immobilization form that is used for the ribozyme of amino acidylate is to be described in Chemistry andBiology 2003; in 10:1077-1084 and the U.S. Patent Application Publication case 2003/0228593, described reference is to be incorporated herein by reference.
The amino process for acylating of chemical includes, but is not limited to by Hecht and colleague (Hecht, S.M.Acc.Chem.Res.1992,25,545; Heckler, T.G.; Roesser, J.R.; Xu, C; Chang, P.; Hecht, S.M.Biochemistry1988,27,7254; Hecht, S.M.; Alford, B.L.; Kuroda, Y.; Kitano, S.J.Biol.Chem.1978,253,4517) and by Schultz, Chamberlin, Dougherty and other 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, people such as J.D., Nature 1992,356, and 537; Gallivan, J.P.; Lester, H.A.; Dougherty, D.A.Chem.Biol.1997,4,740; People such as Turcatti, J.Biol.Chem.1996,271,19991; Nowak, people such as M.W., Science, 1995,268,439; Saks, people such as M.E., J.Biol.Chem.1996,271,23169; Hohsaka, people such as T., J.Am.Chem.Soc.1999,121,34) those methods of avoiding in amino acidylate, using synthetic enzyme of proposing, described reference is to be incorporated herein by reference.The amino process for acylating of described method or other chemical can be used to make the tRNA molecule that amino acidylate takes place.
The method that is used to produce catalytic RNA can relate to the independent set that produces randomization ribozyme sequence, carry out orthogenesis, screens described set and select to show the sequence of those ribozymes of desired aminoacylation activity for desired aminoacylation activity at described set.
Ribozyme can include and is beneficial to active motif of acidylate and/or zone, such as, GGU motif and U rich region.For example, reported that the U rich region can help the identification of amino acid substrate, and the GGU motif can form base pair with 3 of tRNA ' end.In when combination, identification when GGU motif and U rich region help amino acid and tRNA simultaneously, and and then help the amino acidylate of 3 of tRNA ' end.
Ribozyme can be by using and tRNA Asn CCCGThe r24mini of bonded incomplete randomization in vitro selects, and then the systems engineeringization by the concensus sequence of existence among the activity clone produces.The exemplary ribozyme that is obtained by this method is called as " Fx3 ribozyme " and is described in No. the 2003/0228593rd, the open application case of the U.S.; the content of described application case is to be incorporated herein by reference, and described ribozyme serves as and is used for the synthetic multi-usage catalyzer that loads the various aminoacyl-tRNAs of homology alpha-non-natural amino acid.
Immobilization on the substrate can be used to make through effective affinity purification of the tRNA of amino acidylate and can realize.The example of suitable substrate includes, but is not limited to agarose, sepharose and magnetic beads.Ribozyme can be fixed in by the chemical structure of utilizing RNA on the resin, such as 3 on the ribose of RNA '-the cis glycol can produce corresponding dialdehyde through periodate oxidation to help RNA fixing on resin.Can use various types of resins, comprise cheap hydrazides resin, wherein reductive amination makes the interaction between resin and the ribozyme become irreversible key.The synthetic of aminoacyl-tRNA can promote greatly by amino acidylate technology on this post.(Methods 2005 for people such as Kourouklis; 36:239-4) a kind of amino acidylate system based on post is described.
But separation accomplished in various ways through the tRNA of amino acidylate.A kind of suitable method be utilize damping fluid (such as, have 10mM EDTA sodium acetate solution, contain the damping fluid of 50mM N-(2-hydroxyethyl) piperazine-N '-(3-propane sulfonic acid), 12.5mM KCl (pH 7.0), 10mM EDTA or only for edta buffer water (pH7.0)) from the tRNA of pillar wash-out through amino acidylate.
TRNA through amino acidylate can be joined in the translation reaction so that incorporate amino acid into, tRNA is by described amino amino acidylate on the select location in the polypeptide that is made by translation reaction.Can use the example of the translation system of the tRNA through amino acidylate of the present invention to include, but is not limited to cellular lysate.Cellular lysate is provided as the necessary reactive component of in vitro translation that carries out polypeptide from input mRNA.The example of described reactive component includes, but is not limited to ribosomal protein, rRNA, amino acid, tRNA, GTP, ATP, translation initiation factor and elongation factor and other factor relevant with translation.In addition, translation system can be batch formula translation or subregion translation (compartmentalizedtranslation).Batch formula translation system composite reaction component in single compartment, and the subregion translation system is separated translation reaction component and the reaction product that can suppress to translate effect.Described translation system can be buied.
In addition, can use coupling to transcribe/translation system.Coupling transcribes/and translation system allows to import DNA and is transcribed into corresponding mRNA, and this mRNA is translated by reactive component again.The coupling that can buy transcribes/and an example of translation system is quick translation system (Rapid Translation System, RTS, Roche Inc.).Described system comprises containing the mixture that provides such as the intestinal bacteria lysate of the translation components of rrna and translation factor is provided.In addition, comprise that RNA polymerase is to be used for that input DNA is transcribed into the mRNA template that is used to translate.RTS can adopt the subregion translation by placing film between the reaction compartments (comprise supply/consumption compartment and transcribe/translate compartment) that reactive component is separated.
The amino acidylate of tRNA can be carried out by other reagent that includes, but is not limited to transferring enzyme, polysaccharase, catalytic antibody, multifunctional protein etc.
People such as Lu are in Mol Cell.2001 October; 8 (4): describe a kind of method that protein is connected (expressing protein connection) with the synthetic chemistry of peptides that contains alpha-non-natural amino acid among the 759-69.
Also used microinjection technique that alpha-non-natural amino acid is incorporated in the protein.Referring to, for example, 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).Inject xenopus leavis oocytes (Xenopus oocyte) jointly with following two kinds of RNA materials that in vitro make: be coded in the amino acid position place that is paid close attention to have the UAG terminator codon target protein mRNA and suppress tRNA through the amber of the amino acidylate of desired alpha-non-natural amino acid.Then, the machine translator of ovocyte is being inserted alpha-non-natural amino acid by the specified position of UAG.This method has made that generally not meeting the in vitro proteic in vivo structure-functional study of conformity membrane of expression system is carried out.Example comprises incorporates in tachykinin neurokinin-2 acceptor fluorescence amino acid to pass through the FRET (fluorescence resonance energy transfer) measuring distance into, referring to, for example, 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); Incorporate biotinylation amino acid into the surperficial exposed residue in the discriminating ionic channel, referring to, for example, J.P.Gallivan, H.A.Lester and D.A.Dougherty, Chem.Biol., 4:739 (1997); Use cage type tyrosine analogue with the conformational change in the real-time monitoring ionic channel, referring to, for example, J.C.Miller, S.K.Silverman, P.M.England, D.A.Dougherty and H.A.Lester, Neuron, 20:619 (1998); Be used to survey its door control mechanism with use α hydroxy-amino-acid to change the ionic channel framework.Referring to, for example, P.M.England, Y. Zhang, D.A.Dougherty and H.A.Lester, Cell, 96:89 (1999); With T.Lu, A.Y. Ting, J.Mainland, L.Y.Jan, P.G. Schultz and J.Yang, Nat.Neurosci., 4:239 (2001).
In vivo the ability of alpha-non-natural amino acid directly being incorporated in the protein provides the multiple advantage that includes, but is not limited to following advantage: the high yield of mutain, technology simplification, in cell or may study the possibility and the use of these mutains in therapeutic treatment and diagnostic uses of mutain in live organism.Make have various size, the alpha-non-natural amino acid of acidity, nucleophilicity, hydrophobicity and other character is included in ability in the protein and can enlarges our the reasonably and systematically ability of the structure of operon protein widely, to survey novel protein or the organism that protein function and generation have novel character.
Incorporate in the once trial of P-fluoropnenylalanine on site specific ground, the yeast amber is suppressed tRNAPheCUA/ phenylalanyl-tRNA synthetic enzyme to being used for P-fluoropnenylalanine resistance, phenylalanine auxotroph bacillus coli bacterial strain.Referring to, for example, R.Furter, Protein Sci.,7:419 (1998).
Also might use acellular (in vitro) translation system to obtain GH of the present invention (for example, the hGH) expression of polynucleotide.Translation system can be cell system or cell free system, and can be prokaryotic system or eukaryotic system.The cell translation system includes, but is not limited to full cell preparation or the cell culture such as the permeability cell, wherein desired nucleotide sequence can be transcribed into mRNA and mRNA is translated.Cell free translation system is that different types that can buy and many and system are known.The example of cell free system includes, but is not limited to such as the prokaryotic organism lysate of bacillus coli lysate with such as the eukaryote lysate of wheat malt germ extract, insect cell lysate, rabbit skein cell lysate, rabbit oocyte lysate and human cell's lysate.When gained protein through glycosylation, phosphorylation or when other mode was modified, eukaryote extract or lysate can be preferably, this only is possible in eukaryotic system because of many described modifications.In these extracts and the lysate some are (the Promega that can buy; Madison, Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, I11.; GIBCO/BRL; Grand Island, N.Y.).Film extract such as the dog pancreatic extract that contains microsomal membrane also is an available, and it can be used for translating secretory protein.In can comprising that mRNA is as template (in vitro translation) or DNA these systems as template (in vitro transcribing and translating of combination), in vitro synthetic is to guide by rrna.Exploitation cell-free protein expression system has been made considerable effort.Referring to, for example, 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, BiotechnologyProgress, 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); United States Patent (USP) the 6th, 337, No. 191; No. the 2002/0081660th, U.S. Patent Publication case; WO00/55353; WO 90/05785, and it is to be incorporated herein by reference.Can be applied to express and comprise non-naturally encoded amino acid whose GH (for example, hGH) the another kind of method of polypeptide comprises mRNA-peptide integration technology.Referring to, for example, R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 94:12297-12302 (1997); People such as A.Frankel, Chemistry ﹠amp; Biology 10:1043-1050 (2003).In this method, the mRNA template that is connected with tetracycline is translated into peptide on rrna.If modified one or more tRNA molecule so also can be incorporated alpha-non-natural amino acid in the peptide into.After reading last mRNA codon, tetracycline is captured the C end of peptide.Have noticeable character if find the mRNA-peptide binding substances of gained in vitro in the calibrating, its characteristic can easily demonstrate from the mRNA sequence so.Therefore, the those skilled in the art can screen and comprise one or more non-naturally encoded amino acid whose GH (for example, the hGH) storehouse of polypeptide polypeptide of having the character of being wanted with discriminating.Be reported that in the recent period carrying out the translation of rrna in vitro with purified component makes the peptide through non-naturally encoded aminoacid replacement to synthesize.Referring to, for example, people such as A.Forster, Proc.Natl Acad.Sci. (USA) 100:6353 (2003).
Also can use the reconstruct translation system.The lysate that the purified translation factor of the mixture of purified translation factor and warp such as initiation factor-1 (IF-1), IF-2, IF-3 (α or β), EF-T (EF-Tu) or terminator factor replenishes or the combination of lysate also successfully have been used for mRNA is translated as protein.Cell free system also can be that coupling is transcribed/translation system, wherein as Current Protocols in Molecular Biology (editor such as F.M.Ausubel, Wiley Interscience, 1993) described in in the DNA drawing-in system, be transcribed into mRNA and the translation mRNA, described reference is incorporated into thus by reference especially.The RNA that in the eukaryotic transcription system, transcribes can be heteronuclear RNA (hnRNA) or 5 '-end cap (7-methyl guanosine) and 3 '-end poly A has the ripe mRNA form of tail, this can be favourable condition in some translation system.For example, adding cap mRNA is translated in skein cell lysate system with high-level efficiency.
IX. with GH (for example, the hGH) high polymer of polypeptide coupling
The described herein various modifications to non-natural amino acid polypeptides can use described composition, method, technology and strategy to realize herein.These modifications comprise that another functional group that will include, but is not limited to following material incorporates on the alpha-non-natural amino acid component of polypeptide: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; The photocrosslinking agent; Radionuclide; Cytotoxin compounds; Medicine; Affinity labelling; Photoaffinity labeling; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; The antisense polynucleotide; Carbohydrate; Water-soluble branch-shape polymer; Cyclodextrin; Inhibition Yeast Nucleic Acid; Biomaterial; Nanoparticle; Spin labeling; Fluorophore; Metallic part; Radioactive segment; Novel functional group; Covalently or non-covalentlyly with the group of other interaction of molecules; Light cage latching segment); But actinic radiation excitation portion; The intramolecular photosensitization part; Vitamin H; The derivative of vitamin H; The vitamin H analogue; The part of incorporating heavy atom into; But the group of chemical cracking; But the group of photo-cleavage; The side chain of elongation; Sugar through the carbon connection; Redox active agent; Amino thioic acid sulfoacid; Toxic moiety; Through isotope-labeled part; The biophysics probe; Phosphorescent group; Chemiluminescent groups; The intensive group of electronics; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable mark; Small molecules; Quantum dot; The nanometer transmitter; The radioactive nuleus thuja acid; The radioactivity transmitter; Neutron capture agent; Or any combination of above-mentioned substance, or any other desired compound or material.An illustrative, limiting examples as described composition herein, method, technology and strategy, below describe to concentrate on high polymer is added in the non-natural amino acid polypeptides, condition is also to be applicable to (when being necessary at its described composition, method, technology and strategy, suitably modify and for it in addition, the those skilled in the art can utilize disclosure herein to carry out) add other functionality material, it includes, but is not limited to above listed functionality material.
Multiple high polymer and other molecule can be connected to GH of the present invention (for example, hGH) polypeptide to regulate GH (for example, the hGH) biological property of polypeptide, and/or be that (for example, hGH) molecule provides new biological property to GH.These high polymers can be by natural amino acids coding, non-naturally encoded amino acid or natural or alpha-non-natural amino acid any functionality substituting group or be added into any substituting group of natural or alpha-non-natural amino acid or functional group and (for example, hGH) polypeptide is connected with GH.The molecular weight of described polymkeric substance can be in the broad range, includes, but is not limited to be in about 100Da and about 100,000Da or above between.The molecular weight of described polymkeric substance can be and is in about 100Da and about 100, between the 000Da, includes, but is 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 described polymkeric substance can be and is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of described polymkeric substance can be and is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of described polymkeric substance can be and is in approximately 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of described polymkeric substance can be and is in approximately 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of described polymkeric substance can be and is in approximately 10, and 000Da and 40 is between the 000Da.
The invention provides polymkeric substance: the preparation of the homogeneous substantially of protein conjugate." homogeneous substantially " means through observing polymkeric substance as used in this article: the protein conjugate molecule is greater than half of gross protein.Polymkeric substance: protein conjugate has biologic activity and the present invention of being provided herein " homogeneous substantially " PEGization GH (for example, hGH) polypeptide formulations is to be enough homogeneous those preparations with the advantage that represents homogeneous preparation, for example is easy to predict the pharmacokinetics character of between-lot in clinical application.
The those skilled in the art also can select to prepare polymkeric substance: the mixture of protein conjugate molecule, and the advantage that is provided is herein, and the those skilled in the art can select single polymkeric substance: protein conjugate is included in the ratio in the mixture.Therefore, if when being necessary, the those skilled in the art can prepare have the various quantity that connected polymer moieties (promptly, two-, three-, four-etc.) the mixture of range protein, and single polymkeric substance: protein conjugate combination, and the single polymkeric substance that obtains having predetermined proportion: the mixture of protein conjugate with described binding substances and use method preparation of the present invention.
Selected polymkeric substance can be water miscible, so that connected protein can not precipitated in aqueous environments (such as physiological environment).This polymkeric substance can be branched or not branched.For the therepic use of final product preparation, this polymkeric substance will be for pharmaceutically acceptable.
The example of polymkeric substance (for example includes, but is not limited to poly alkyl ether and its alkoxy end-capped analogue, polyoxyethylene glycol, polyoxyethylene/propylene glycol and its methoxy or ethoxy end-blocking analogue, especially be polyoxyethylene glycol, the latter is also referred to as polyoxyethylene glycol or PEG); Polyvinylpyrrolidone; The polyvinyl alkyl oxide; The Ju oxazoline; Ju Wan oxazolin and poly-Qiang Wan oxazolin; Polyacrylamide, poly-alkyl acrylamide and poly-hydroxyalkyl acrylamide (for example poly-hydroxypropylmethyl acrylamide and its derivative); Poly-hydroxyalkyl acrylates; Polysialic acid and its analogue; The hydrophilic peptide sequence; Polysaccharide and its derivative comprise dextran and glucan derivative, for example Sensor Chip CM 5, dextran sulfate, glycosaminoglycan; Mierocrystalline cellulose and its derivative, for example carboxymethyl cellulose, hydroxy alkyl cellulose; Chitin and its derivative, for example chitosan, succinyl chitosan, carboxymethyl chitin, carboxymethyl chitosan; Hyaluronic acid and its derivative; Starch; Alginates; Chondroitin sulfate; Albumin; Amylopectin and carboxymethyl amylopectin; Polyamino acid and its derivative, for example polyglutamic acid, polylysine, poly-aspartic-acid, poly-asparagine; Maleic anhydride copolymer, such as: styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer; Polyvinyl alcohol; Its multipolymer; Its trimer; Its mixture; Derivative with aforementioned polymer.
The ratio of peg molecule and protein molecule will change, and this just will change as its concentration in reaction mixture.Generally speaking, best ratio (with regard to the efficient of the reaction that has minimum excessive unreacted protein or polymkeric substance) can be determined by the molecular weight of selected polyoxyethylene glycol and the useful number of available reactive group.When relating to molecular weight, the molecular weight of polymkeric substance is high more usually, and the quantity of the polymer molecule that can be connected with protein is just few more.Similarly, when optimizing these parameters, should consider the branch of polymkeric substance.Usually, molecular weight high more (or branch is many more), polymkeric substance: protein rate is just high more.
As used herein, and when containing PEG:GH (for example hGH) polypeptide conjugates, term " treatment significant quantity " refers to the amount that the benefit of wanting is provided for the patient.This amount will change according to individual difference, and will decide on some factors (comprising patient's the overall physical appearance and the potential cause of disease of illness to be treated).(for example, hGH) amount of polypeptide provides acceptable velocity of variation and desired reaction is maintained useful level the GH that is used for the treatment of.The treatment significant quantity of the present composition can use open available material and program easily to determine by the those skilled in the art.
Water-soluble polymers can be any structure form, includes, but is not limited to straight chain, bifurcated or branch.Usually, water-soluble polymers such as poly-(ethylene glycol) (PEG), but also can adopt other water-soluble polymers for poly-(alkylene glycols).For example, PEG is used to describe some embodiment of the present invention.
PEG is known water-soluble polymers, it is commercially available or can be according to the known method of those skilled in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, the 3rd volume, the 138th to 161 page), prepare by the ring-opening polymerization of ethylene glycol.Term " PEG " is widely used for containing any peg molecule, does not consider size or the terminal modification of PEG, and can by following formula be expressed as with GH (for example, hGH) polypeptide connects:
XO-(CH 2CH 2O) n-CH 2CH 2-Y.
Wherein n be 2 to 10,000 and X be H or include, but is not limited to C 1-4Alkyl, protecting group or functional end-group end modified.
In some cases, used PEG is termination with hydroxyl or methoxyl group on an end among the present invention, and promptly X is H or CH 3(" methoxyl group PEG ").Perhaps, PEG can reactive group be to stop, and forms the double functional copolymer thus.The type reaction group can comprise those be usually used in 20 kinds of common amino acids in the reactive group of the functional group reactions that exists (include, but is not limited to maleimide groups, activated carbonic ether (including, but is not limited to the p-nitrophenyl ester), activated ester (includes, but is not limited to N-maloyl imines, the p-nitrophenyl ester) and aldehyde) and for 20 kinds of common amino acids be inertia but be present in the functional group that complementary functional groups in the non-naturally encoded amino acid plays specific reaction and (include, but is not limited to azido-, alkynyl).The other end that it should be noted that the PEG that represents with Y in following formula will directly or indirectly be connected in GH (for example, hGH) polypeptide via naturally occurring amino acid or non-naturally encoded amino acid.For example, Y can be amido linkage, amino-formate bond or the urea key of the amido (including, but is not limited to the ε amine or the N-end of Methionin) that is connected to polypeptide.Perhaps, Y can be the maleimide key that is connected to thiol group (including, but is not limited to the thiol group of halfcystine).Perhaps, Y can be and is connected to common key via 20 kinds of unapproachable residues of common amino acid.For example, (for example, the hGH) reaction of the alkynyl on the polypeptide is to form Hu Shi root [3+2] cycloaddition product can to make azido-on the PEG and GH.Perhaps, can make alkynyl and the azido-reaction that is present in the non-naturally encoded amino acid on the PEG, to form similar product.In certain embodiments, can be when suitable, make strong nucleophile (including, but is not limited to hydrazine, hydrazides, azanol, Urea,amino-) and the aldehyde radical or the ketone group reaction that are present in the non-naturally encoded amino acid, to form hydrazone, oxime or semicarbazone, it in some cases can be by handling and further reduction with suitable reductive agent.Perhaps, strong nucleophile can be incorporated GH into by non-naturally encoded amino acid and (for example, hGH) in the polypeptide, and is used for preferentially and ketone group that is present in water-soluble polymers or aldehyde radical reaction.
Any molecular mass of PEG can be used by actual needs, and it includes, but is not limited to about 100 dalton (Da) as required to 100,000Da or above (including, but is not limited to 0.1 to 50kDa or 10 sometimes to 40kDa).The molecular weight of PEG can be in the broad range, and it includes, but is not limited to be in about 100Da and about 100,000Da or above between.The molecular weight of PEG can be and is in about 100Da and about 100, between the 000Da, includes, but is 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, 35,000 Da, 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 PEG is for being in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of PEG is for being in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of PEG is about 1 for being in, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 5 for being in, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 10 for being in, and 000Da and 40 is between the 000Da.Also can use side chain PEG, it includes, but is not limited to the PEG molecule that each chain wherein has the molecular weight that (includes, but is not limited to 1 to 50kDa or 5 to 20kDa) in 1 to the 100kDa scope.The molecular weight of each chain of side chain PEG can be (including, but is not limited to) be in about 1,000Da and about 100,000Da or above between.The molecular weight of each chain of side chain PEG can be and is in approximately 1, and 000Da and about 100 between the 000Da, includes, but is not limited to 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 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, the molecular weight of each chain of side chain PEG is about 1 for being in, and 000Da and 50 is between the 000Da.In certain embodiments, the molecular weight of each chain of side chain PEG is about 1 for being in, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of each chain of side chain PEG is about 5 for being in, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of each chain of side chain PEG is about 5 for being in, and 000Da and 20 is between the 000Da.At (including, but is not limited to) Shearwater Polymers, the PEG molecule of broad range is described among the Inc.catalog, Nektar Therapeutics catalog (it is incorporated herein by reference).
Generally speaking, at least one end of PEG molecule can be used for and the reaction of non-naturally encoded amino acid.For example, having the alkynyl and the azido-PEG derivative partly that are suitable for the amino acid side chain reaction can be used for making PEG to be connected in non-naturally encoded as described herein amino acid.If non-naturally encoded amino acid comprises the nitrine part, PEG will contain the alkynyl part usually with formation [3+2] cycloaddition product so, or for containing the basic activated PEG material (that is, ester, carbonic ether) of seeing to form amido linkage.Perhaps, if non-naturally encoded amino acid comprises alkynyl moiety, PEG will contain the nitrine part usually to form [3+2] Hu Shi root cycloaddition product so.If non-naturally encoded amino acid comprises carbonyl, PEG will comprise effective nucleophile (including, but is not limited to hydrazides, hydrazine, azanol, Urea,amino-functional group) usually so, so that form corresponding hydrazone key, oxime key and semicarbazone key respectively.In other alternate embodiment, can use the oriented opposite group of above-mentioned reactive group, can make azido-part and the PEG derivatives reaction that contains alkynyl in the non-naturally encoded amino acid.
In certain embodiments, have the PEG derivative GH (for example, hGH) polypeptide variants contain can with the chemical functional group who is present in the chemical functional group reaction on the non-naturally encoded amino acid side chain.
The present invention provides the polymer derivant that contains azido-and the polymer derivant that contains ethynyl in certain embodiments, and it comprises, and to have about 800Da extremely about 100, the water-soluble polymers main chain of the molecular-weight average of 000Da.The main polymer chain of this water-soluble polymers can be poly-(ethylene glycol).Yet, should be appreciated that, multiple water-soluble polymers (include, but is not limited to polyoxyethylene glycol and other related polymer, comprise poly-(dextran) and poly-(propylene glycol)) also is applicable to practice of the present invention, and all these molecules are contained and comprised in the use expection of term PEG or poly-(ethylene glycol).Term PEG includes, but is not limited to any type of poly-(ethylene glycol), comprises difunctionality PEG, multi-arm PEG, derivatize PEG, bifurcated PEG, branch PEG, side joint PEG (promptly having PEG or the related polymer of one or more side joints in the functional group of main polymer chain) or wherein has the PEG of degradable key.
That PEG is generally is transparent, colourless, do not have smell, water soluble, heat stable, to many chemical reagent be inertia, can not hydrolysis or rotten and be generally nontoxic.Poly-(ethylene glycol) is considered to be biocompatible, that is to say that PEG can coexist and can not cause damage with living tissue or organism.More particularly, PEG is a non-immunogenic substantially, that is to say that PEG is not inclined to produce immune response in vivo.When being connected in the molecule (such as biologically active agent) with some ideal functionalities in vivo, PEG tends to shelter this reagent and can reduce or eliminate any immune response, so that make the existence of this reagent of organism tolerable.The PEG binding substances can not tend to produce the immune response of essence or cause condense or other ill effect.Has formula-CH 2CH 2O-(CH 2CH 2O) n--CH 2CH 2-PEG (wherein n is about 3 to about 4000, is generally about 20 to about 2000) be applicable to the present invention.Have about 800Da to about 100, the PEG of 000Da molecular weight is particularly useful as main polymer chain in some embodiments of the invention.The molecular weight of PEG can be in the broad range, and it includes, but is not limited to be in about 100Da and about 100,000Da or above between.The molecular weight of PEG can be and is in about 100Da and about 100, between the 000Da, includes, but is 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 PEG is for being in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of PEG is for being in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of PEG is about 1 for being in, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 5 for being in, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 10 for being in, and 000Da and 40 is between the 000Da.
Main polymer chain can be straight chain or branched.The branched polymers main chain is generally known in affiliated field.Usually, branched polymers has branch core, a center and a plurality of straight-chain polymer chain that is connected to this center branch core.PEG uses with the branch form, and this form can prepare by the addition of ethylene oxide to multiple polyvalent alcohol (such as glycerine, glycerin oligomer, tetramethylolmethane and sorbyl alcohol).Center branch part also can be derived by some seed amino acids (such as Methionin) and be obtained.Branch poly-(ethylene glycol) can be by general formula R (PEG-OH) mExpression, wherein R is derived from core (such as glycerine, glycerin oligomer or tetramethylolmethane), and m represents the arm number.Such as United States Patent (USP) the 5th, 932, No. 462; The 5th, 643, No. 575; The 5th, 229, No. 490; The 4th, 289, No. 872; U.S. patent application case 2003/0143596; WO 96/21469; Also can be used as main polymer chain with the multi-arm PEG molecule of those PEG molecules described in the WO 93/21259 (its each person's full text is incorporated herein by reference).
Branch PEG also can for by PEG (--YCHZ 2) nThe bifurcated PEG form of expression, wherein Y is a linking group, and Z is the activation end group that is connected with CH by a series of atom with designated length.
Another kind of branch form, i.e. side joint PEG is along the PEG main chain but not have reactive group such as carboxyl on the PEG chain end.
Except these PEG forms, polymkeric substance also can have weak or degradable key through preparation in main chain.For example, PEG can have the ester bond of the hydrolysis of being easy to through preparation in main polymer chain.As follows, this hydrolytic action can cause polymer cracking to become to have the fragment of lower molecular weight:
-PEG-CO 2-PEG-+H 2O→PEG-CO 2H+HO-PEG-
The those skilled in the art understands, and term poly-(ethylene glycol) or PEG represent or comprise known form of ownership in the affiliated field, those forms that it includes, but is not limited to disclose herein.
Many other polymkeric substance also are applicable to the present invention.In certain embodiments, have 2 water-soluble polymers main chains and be particularly useful for the present invention to about 300 ends.The example of suitable polymers includes, but is not limited to other poly-(alkylene glycols) such as poly-(propylene glycol) (" PPG "), its multipolymer (including, but is not limited to the multipolymer of ethylene glycol and propylene glycol), its trimer, its mixture etc.Although the molecular weight of each chain can change in the main polymer chain, its usually at about 800Da to about 100, in the scope of 000Da, often be about 6,000Da is extremely about 80, in the scope of 000Da.The molecular weight of each chain can be and is in about 100Da and about 100 in the main polymer chain, and between the 000Da, it includes, but is 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 is for being in about 100Da and 50, between the 000Da in the main polymer chain.In certain embodiments, the molecular weight of each chain is for being in about 100Da and 40, between the 000Da in the main polymer chain.In certain embodiments, the molecular weight of each chain is about 1 for being in the main polymer chain, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of each chain is about 5 for being in the main polymer chain, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of each chain is about 10 for being in the main polymer chain, and 000Da and 40 is between the 000Da.
Those skilled in the art will realize that the aforementioned list of water-soluble main chain is exhaustive by no means substantially, and only be illustrative, and all polymeric materials that will have an above-mentioned characteristic all are considered as being applicable to the present invention.
In some embodiments of the invention, polymer derivant is " multi-functional ", mean main polymer chain and have at least two, and may be functionalized or activatory is terminal through functional group up to about 300.Multifunctional polymer derivant includes, but is not limited to have the straight-chain polymer of two ends, and wherein each terminal bond is to being identical or different functional group.
In certain embodiments, polymer derivant has following structure:
X-A-POLY-B-N＝N＝N，
Wherein:
N=N=N is the azido-part;
B is the connection portion, and it can exist or not exist;
POLY is water-soluble nonantigenic polymkeric substance;
A is the connection portion, and it can exist or not exist, and it can be identical or different with B; And
X is second functional group.
The example of connection portion A and B includes, but is not limited to contain at the most 18 and may contain the multiple functionalised alkyl of 1 to 10 carbon atom.Can comprise heteroatoms in the alkyl chain, such as nitrogen, oxygen or sulphur.Alkyl chain also can be at heteroatoms punishment branch.Other example of connection portion A and B includes, but is not limited to contain at the most 10 and may contain the multiple functionalized aryl of 5 to 6 carbon atoms.This aryl can replace through another carbon atom, nitrogen-atoms, Sauerstoffatom or sulphur atom.Other example of suitable linking group comprises those at United States Patent (USP) the 5th, 932, No. 462, the 5th, 643, and No. 575; With the linking group described in the U.S. Patent Application Publication case 2003/0143596 (it respectively is incorporated herein by reference).The aforementioned list that those skilled in the art will realize that the connection portion is exhaustive absolutely not, and only is illustrative, and all polymeric materials that will have an above-mentioned characteristic all are considered as being applicable to the present invention.
The example that is suitable for use as the functional group of X includes, but is not limited to hydroxyl; through the protection hydroxyl; alkoxyl group; active ester (such as N-maloyl imido grpup ester and 1-benzotriazole base ester); activated carbon acid esters (such as N-maloyl imido grpup carbonic ether and 1-benzotriazole base carbonic ether); acetal; aldehyde; aldehydrol; thiazolinyl; acrylate; methacrylic ester; acrylamide; active sulfone; amine; the amino oxygen base; through protection amine; hydrazides; through the protection hydrazides; through protection mercaptan; carboxylic acid; through the protection carboxylic acid; isocyanic ester; lsothiocyanates; maleimide; vinyl sulfone(Remzaol; two thiopyridines; vinyl pyridine; iodo-acid amide; epoxide; oxalic dialdehyde; diketone; methanesulfonates; tosylate; the trifluoro esilate; alkene; ketone and trinitride functional group.As be appreciated by one of skill in the art that selected X part should be compatible with azido-, in order to avoid react with azido-.The polymer derivant that contains azido-is same difunctionality (homobifunctional), and this means second functional group (being X) and also is the azido-part; Or be Heterobifunctional, this means second functional group is different functional groups.
Term " through protection " refers to protecting group or the existence partly that prevents that chemical reactivity functional group from reacting under some reaction conditions.Protecting group will be looked the type of the chemically reactive group of being protected and be changed.For example, if chemically reactive group is amine or hydrazides, protecting group can be selected from the group of tertbutyloxycarbonyl (t-Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc) so.If chemically reactive group is a mercaptan, protecting group can be adjacent pyridyl disulfide so.If chemically reactive group is carboxylic acid (such as butyric acid or propionic acid) or hydroxyl, protecting group can be benzyl or alkyl (such as methyl, ethyl or the tertiary butyl) so.Known other protecting group also can be used for the present invention in the affiliated field.
The particular instance of functional end-group includes, but is not limited to N-succimide base carbonic ether (referring to for example United States Patent (USP) the 5th in the document, 281, No. 698, the 5th, 468, No. 478), amine (referring to people Makromol.Chem.182:1379 (1981) such as for example Buckmann, people Eur.Polym.J.19:1177 (1983) such as Zalipsky), hydrazides (referring to people Makromol.Chem.179:301 (1978) such as for example Andresz), succimide base propionic ester and succimide base butyric ester are (referring to people such as for example Olson in Poly (ethylene glycol) Chemistry ﹠amp; Biological Applications, the 170th to 181 page, Harris ﹠amp; Zalipsky Eds., ACS, Washington, D.C., 1997; Also referring to United States Patent (USP) the 5th, 672, No. 662), succimide base succinate (referring to people Makromol.Chem.180:1381 (1979) such as people Cancer Biochem.Biophys.7:175 (1984) such as for example Abuchowski and Joppich), the succimide ester is (referring to for example United States Patent (USP) the 4th, 670, No. 417), the benzotriazole carbonic ether is (referring to for example United States Patent (USP) the 5th, 650, No. 234), glycidyl ether is (referring to people Eur.J Biochem.94:11 (1979) such as for example Pitha, Elling et al, Biotech.Appl.Biochem.13:354 (1991)), the oxygen carbonylic imidazole is (referring to people such as for example Beauchamp, Anal.Biochem.131:25 (1983), people J.Controlled Release 1:251 (1985) such as Tondelli), the p-nitrophenyl carbonic ether is (referring to people such as for example Veronese, Appl.Biochem.Biotech., 11:141 (1985); With people such as Sartore, Appl.Biochem.Biotech., 27:45 (1991)), aldehyde is (referring to people J.Polym.Sci.Chem.Ed.22:341 (1984) such as for example Harris, United States Patent (USP) the 5th, 824, No. 784, United States Patent (USP) the 5th, 252, No. 714), maleimide is (referring to people Biotechnology (NY) 8:343 (1990) such as for example Goodson, Romani et al.in Chemistry of Peptides and Proteins2:29 (1984) and Kogan, Synthetic Comm.22:2417 (1992)), adjacent pyridyl disulfide (referring to people Bioconj.Chem.4:314 (1993) such as for example Woghiren), vinylcarbinol is (referring to people Macromolecules such as for example Sawhney, 26:581 (1993)), vinyl sulfone(Remzaol is (referring to for example United States Patent (USP) the 5th, 900, No. 461).All above-mentioned documents and patent all are incorporated herein by reference.
In certain embodiments of the invention, polymer derivant of the present invention comprises the main polymer chain with following structure:
X-CH 2CH 2O--(CH 2CH 2O) n--CH 2CH 2-N＝N＝N，
Wherein:
X is aforesaid functional group; And
N is about 20 to about 4000.
In another embodiment, polymer derivant of the present invention comprises the main polymer chain with following structure:
X-CH 2CH 2O--(CH 2CH 2O) n--CH 2CH 2-O-(CH 2) m-W-N＝N＝N，
Wherein:
W is aliphatic series or the aromatics connexon part that comprises 1 to 10 carbon atom;
N is about 20 to about 4000; And
X is aforesaid functional group, and m is between 1 and 10.
The PEG derivative that contains azido-of the present invention can be by several different methods preparation known and/or disclosed herein in the affiliated field.In a kind of method shown below, make to have about 800Da to about 100, the water-soluble polymers main chain of 000Da molecular-weight average, first end with bond to the first functional group and bond to the main polymer chain of second end of suitable leavings group and azido-negatively charged ion (its can with any ion pairing in some suitable counter (comprising sodium ion, potassium ion, tertiary butyl ammonium ion etc.)) reacts.This leavings group stands nucleophilic displacement and is partly replaced by azido-, obtains containing the PEG polymkeric substance of azido-.
X-PEG-L+N 3 -→X-PEG-N 3
As shown in it, be used for suitable polymers main chain of the present invention and have formula X-PEG-L, wherein PEG is poly-(ethylene glycol), and X be the functional group of not reacting with azido-, and L is suitable leavings group.The example of appropriate functional group includes, but is not limited to hydroxyl, through protection hydroxyl, acetal, thiazolinyl, amine, amino oxygen base, through protection amine, through the protection hydrazides, through protection mercaptan, carboxylic acid, through protection carboxylic acid, maleimide, two thiopyridines and vinyl pyridine and ketone.The example of suitable leavings group includes, but is not limited to muriate, bromide, iodide, methanesulfonates, trifluoro esilate and toluenesulphonic acids ester group.
In the another kind of preparation method of the polymer derivant that contains azido-of the present invention, make linking agent and have about 800Da to about 100 with azido-functional group, the water-soluble polymers main chain contact of 000Da molecular-weight average, wherein this linking agent has functional group, its will be optionally with the PEG polymkeric substance on chemical functional group's reaction, contain the polymer derivant product of azido-with formation, wherein azido-separates by linking group and main polymer chain.
Hereinafter show exemplary reaction process:
X-PEG-M+N-connexon-N=N=N → PG-X-PEG-connexon-N=N=N,
Wherein:
PEG is poly-(ethylene glycol), and X is capping group, such as alkoxyl group or aforesaid functional group; And
M be not with the azido-functional group reactions but will be effectively and optionally with the functional group of N functional group reactions.
The example of appropriate functional group includes, but is not limited to: if N is an amine, M is carboxylic acid, carbonic ether or active ester so; If N is hydrazides or amino oxygen base section, M is a ketone so; If N is a nucleophile, M is a leavings group so.
The purifying of crude product can be finished by currently known methods, and these methods include, but is not limited to product precipitation, then chromatography where necessary.
Be presented at the more particularly example under the situation of PEG diamines hereinafter, wherein a kind of protected group part in these amine (such as the tertiary butyl-Boc) protection, and make gained list protection PEG diamines and connection portion reaction with azido-functional group:
BocHN-PEG-NH 2+HO 2C-(CH 2) 3-N＝N＝N。
In the case, can use multiple activator (such as thionyl chloride or carbodiimide reagent and N-maloyl imines or N-hydroxybenzotriazole) that amido is coupled to the carboxylic acid group, with at monoamine PEG derivative and have between the connexon part of azido-and form the amine key.After successfully forming the amine key, gained contains the azido-derivative and can be directly used in the modified biological bioactive molecule through the protection of the N-tertiary butyl-Boc-, or it can be through further refining to add other useful functional group.For example, the N-tertbutyloxycarbonyl can be by the hydrolysis with strong acid treatment, to generate ω-amido-PEG-trinitride.Gained amine can be used as synthetic (synthetic handle) to add other useful functional group (such as maleimide groups, activation disulphide, Acibenzolar etc.), in order to form valuable Heterobifunctional reagent.
It is connected to each of polymkeric substance particularly suitable when terminal with differing molecular to the Heterobifunctional derivative at needs.For example, ω-N-amino-N-azido-PEG is connected to the end of PEG with the molecule (such as aldehyde, ketone, Acibenzolar, activated carbonate etc.) that allows to have the activation electrophilic group, and the molecule that allows to have ethynyl is connected to another end of PEG.
In another embodiment of the present invention, polymkeric substance has following structure:
X-A-POLY-B-C≡C-R，
Wherein:
R can be H or alkyl, thiazolinyl, alkoxyl group or aryl or is substituted aryl;
B is the connection portion, and it can exist or not exist;
POLY is water-soluble nonantigenic polymkeric substance;
A is the connection portion, and it can exist or not exist, and it can be identical or different with B; And
X is second functional group.
The example of connection portion A and B includes, but is not limited to contain at the most 18 and may contain the multiple functionalised alkyl of 1 to 10 carbon atom.Can comprise heteroatoms in the alkyl chain, such as nitrogen, oxygen or sulphur.Alkyl chain also can be at heteroatoms punishment branch.Other example of connection portion A and B includes, but is not limited to contain at the most 10 and may contain the multiple functionalized aryl of 5 to 6 carbon atoms.This aryl can replace through another carbon atom, nitrogen-atoms, Sauerstoffatom or sulphur atom.Other example of suitable linking group comprises those at United States Patent (USP) the 5th, 932, No. 462, the 5th, 643, and No. 575; With the linking group described in the U.S. Patent Application Publication case 2003/0143596 (it respectively is incorporated herein by reference).The aforementioned list that those skilled in the art will realize that the connection portion is exhaustive absolutely not, and only is contemplated to illustratively, and the multiple polymeric material that will have an above-mentioned characteristic all is considered as being applicable to the present invention.
The example that is suitable for use as the functional group of X includes, but is not limited to hydroxyl; through the protection hydroxyl; alkoxyl group; active ester (such as N-maloyl imido grpup ester and 1-benzotriazole base ester); activated carbon acid esters (such as N-maloyl imido grpup carbonic ether and 1-benzotriazole base carbonic ether); acetal; aldehyde; aldehydrol; thiazolinyl; acrylate; methacrylic ester; acrylamide; active sulfone; amine; the amino oxygen base; through protection amine; hydrazides; through the protection hydrazides; through protection mercaptan; carboxylic acid; through the protection carboxylic acid; isocyanic ester; lsothiocyanates; maleimide; vinyl sulfone(Remzaol; two thiopyridines; vinyl pyridine; iodo-acid amide; epoxide; oxalic dialdehyde; diketone; methanesulfonates; tosylate; the trifluoro esilate; alkene; ketone and ethynyl.Understand as us, selected X part should be compatible with azido-, in order to avoid react with azido-.The polymer derivant that contains ethynyl is same difunctionality (homobifunctional), means second functional group (being X) and also is the ethynyl part; Or be Heterobifunctional, meaning second functional group is different functional groups.
In another embodiment of the present invention, polymer derivant comprises the main polymer chain with following structure:
X-CH 2CH 2O--(CH 2CH 2O) n--CH 2CH 2-O-(CH 2) m-C≡CH，
Wherein:
X is aforesaid functional group;
N is about 20 to about 4000; And
M is between 1 and 10.
The particular instance that shows each Heterobifunctional PEG polymkeric substance hereinafter.
The PEG derivative that contains ethynyl of the present invention can use those skilled in the art's method known and/or disclosed herein to prepare.In one approach, make to have about 800Da to about 100, the water-soluble polymers main chain of 000Da molecular-weight average, first end and bond with bond to the first functional group to the main polymer chain of second end of suitable nucleophilic group with have ethynyl functional group and leavings group (its be suitable for PEG on the nucleophilic group reaction) the compound reaction.When having nucleophilic PEG polymkeric substance partly and having the molecular combinations of leavings group, this leavings group stands nucleophilic displacement and is partly replaced by nucleophilic, obtains the desired ethynyl polymer that contains.
X-PEG-Nu+I-A-C→X-PEG-Nu-A-C≡CR’
As shown in it, the preferred polymers main chain that is used for this reaction has formula X-PEG-Nu, and wherein PEG is poly-(ethylene glycol), Nu be nucleophilic part and X for not with the functional group of Nu, L or ethynyl functional group reactions.
The example of Nu includes, but is not limited to amine, alkoxyl group, aryloxy, sulfydryl, imino-, carboxylicesters, hydrazides, amino oxygen base, and it mainly reacts via SN2 type mechanism.Other example of Nu group comprises the functional group that those will mainly react via nucleophilic addition.The L examples of groups comprises that muriate, bromide, iodide, methanesulfonates, trifluoro esilate and toluenesulphonic acids ester group and other expection stand the group of nucleophilic displacement, stand the electrophilic group of nucleophile addition as ketone, aldehyde, thioesters, alkene, alpha-beta unsaturated carbonyl, carbonic ether and other expection.
In another embodiment of the present invention, A is the aromatic ring that is substituted of the aliphatic connexon of 1 to 10 carbon atom or 6 to 14 carbon atoms.X be not with the functional group of azido-reaction, and L is suitable leavings group.
In the another kind of preparation method who contains the ethynyl polymer derivative of the present invention; make to have about 800Da, the 000Da molecular-weight average, on an end, have through protection functional group or end-capping reagent and the PEG polymkeric substance that on another end, has a suitable leavings group and contact with the acetylene negatively charged ion to about 100.
Show exemplary reaction process hereinafter:
X-PEG-L+-C≡CR’→X-PEG-C≡CR’
Wherein:
PEG is a capping group for gathering (ethylene glycol) and X, such as alkoxyl group or functional group mentioned above; And
R ' is H, alkyl, alkoxyl group, aryl or aryloxy, or is substituted alkyl, is substituted alkoxyl group, is substituted aryl or is substituted aryloxy.
In above-mentioned example, leavings group L should have enough reactivities, to stand the displacement of SN2 type when contacting with the acetylene negatively charged ion of enough concentration.Finish leavings group through the required reaction conditions of acetylene negatively charged ion SN2 displacement for the those skilled in the art for known.
The purifying of crude product can be finished by known method in the affiliated field usually, and these methods include, but is not limited to product precipitation, then chromatography where necessary.
Water-soluble polymers can be connected to GH of the present invention (for example, hGH) polypeptide.Water-soluble polymers can be via (for example incorporating GH into, hGH) the non-naturally encoded amino acid in the polypeptide, or any functional group or substituting group non-naturally encoded or natural amino acids coding, or join any functional group non-naturally encoded or natural amino acids coding or substituting group and connect.Perhaps, water-soluble polymers is to be connected to via naturally occurring amino acid (including, but is not limited to the amido of halfcystine, Methionin or N-terminal residue) wherein to incorporate non-naturally encoded amino acid whose GH (for example, hGH) polypeptide into.In some cases, GH of the present invention (for example, hGH) polypeptide comprises 1,2,3,4,5,6,7,8,9, the alpha-non-natural amino acid more than 10 or 10, one of them or more than one non-naturally encoded amino acid be connected in water-soluble polymers (including, but is not limited to PEG and/or oligose).In some cases, (for example, hGH) polypeptide comprises 1,2,3,4,5,6,7,8,9, is connected to the natural amino acids coding of water-soluble polymers more than 10 or 10 GH of the present invention in addition.In some cases, (for example, hGH) polypeptide comprises one or more non-naturally encoded amino acid that are connected to water-soluble polymers and one or more and is connected to the naturally occurring amino acid of water-soluble polymers GH of the present invention.In some embodiment kinds, be used for water-soluble polymers of the present invention and for non-binding form, can strengthen GH (for example, the hGH) serum half-life of polypeptide.
Can regulate and (for example be connected to GH of the present invention, hGH) quantity of the water-soluble polymers of polypeptide (being PEGization or glycosylated degree), so that (include, but is not limited to increase or reduce) pharmacological characteristic, pharmacokinetics feature or the pharmacodynamic properties of change to be provided, such as transformation period in vivo.In certain embodiments, GH (for example, hGH) transformation period of polypeptide with respect to increase for modified polypeptide at least about 10%, 20%, 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.
Contain the PEG derivative that strong nucleophilicity base closes (being hydrazides, hydrazine, azanol or Urea,amino-)
In one embodiment of the invention, comprise that the non-naturally encoded amino acid whose GH that contains carbonyl (for example, hGH) modify through the PEG derivative that contains terminal hydrazine, azanol, hydrazides or Urea,amino-part (it is connected directly to the PEG main chain) by polypeptide.
In certain embodiments, the terminal PEG derivative of azanol will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-O-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000 (that is, molecular-weight average be 5 to 40kDa).
In certain embodiments, the PEG derivative that contains hydrazine or contain hydrazides part will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-X-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000, and X is optionally for can exist or non-existent carbonyl (C=O).
In certain embodiments, contain Urea,amino-PEG derivative partly and will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-NH-C(O)-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000.
In another embodiment of the present invention, comprise that the GH that contains carbonylamino acid (for example, hGH) modify through the PEG derivative that contains terminal azanol, hydrazides, hydrazine or Urea,amino-part (it is connected to the PEG main chain via amido linkage) by polypeptide.
In certain embodiments, the terminal PEG derivative of azanol has following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-O-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000 (that is, molecular-weight average be 5 to 40kDa).
In certain embodiments, the PEG derivative that contains hydrazine or contain hydrazides part has following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-X-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2 to 10, and n is 100 to 1,000, and X is optionally for can exist or non-existent carbonyl (C=O).
In certain embodiments, contain Urea,amino-PEG derivative partly and have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-NH-C(O)-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000.
In another embodiment of the present invention, comprise contain carbonylamino acid GH (for example, hGH) polypeptide is modified through the branch PEG derivative that contains terminal hydrazine, azanol, hydrazides or Urea,amino-part, and wherein each chain of this branch PEG has in 10 to the 40kDa scopes and may be the molecular weight in 5 to the 20kDa scopes.
In another embodiment of the present invention, (for example, hGH) the PEG derivative of polypeptide through having apparatus derivatorius modified to comprise non-naturally encoded amino acid whose GH.For example, in certain embodiments, the terminal PEG derivative of hydrazine end or hydrazides will have following array structure:
[RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)] 2CH(CH 2) m-X-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000, and X is optionally for can exist or non-existent carbonyl (C=O).
In certain embodiments, contain Urea,amino-PEG derivative partly and will have following structure:
[RO-(CH 2CH 2O) n-O-(CH 2) 2-C(O)-NH-CH 2-CH 2] 2CH-X-(CH 2) m-NH-C(O)-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X optionally is NH, O, S, C (O) or do not exist, m be 2 to 10 and n be 100 to 1,000.
In certain embodiments, the PEG derivative that contains the azanol group will have following structure:
[RO-(CH 2CH 2O) n-O-(CH 2) 2-C(O)-NH-CH 2-CH 2] 2CH-X-(CH 2) m-O-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X optionally is NH, O, S, C (O) or do not exist, m be 2 to 10 and n be 100 to 1,000.
Water-soluble polymers is connected to the degree and the position of hGH polypeptide can regulate the hGH polypeptide to the combination of hGH polypeptide receptor on position 1.In certain embodiments, the invention provides the GH (for example hGH) that is connected with at least one PEG by the oxime key, the PEG that wherein is used to form the oxime key in this reaction is mono methoxy-poly-(ethylene glycol)-2-amino oxygen base ethamine carbamate hydrochloride of straight chain, 30kDa, as shown in Figure 19.Figure 20 is presented on the synthetic illustrative example of straight chain suitable in synthesizing of specific embodiment of the present invention, the mono methoxy of 30kDa-poly-(ethylene glycol)-2-amino oxygen base ethamine carbamate hydrochloride.
Only for example and not as for described composition herein, method, the PEG types of agents that technology and strategy together use or the restriction of kind, Figure 21 presents other illustrative example of containing azanol PEG reagent (these PEG reagent can contain the oxime non-natural amino acid polypeptides with what formation was connected to the PEG group with the non-natural amino acid polypeptides reaction that contains carbonyl) and contains the example of carbonyl PEG reagent (these PEG reagent can with contain the oxime non-natural amino acid polypeptides or contain the reaction of azanol non-natural amino acid polypeptides is connected to the PEG group with formation the new oxime non-natural amino acid polypeptides that contains).Figure 22 present be used to form contain azanol PEG reagent or contain azanol PEG reagent through the protection form or contain four illustrative example through the synthetic method of the form of sheltering of azanol PEG reagent.Figure 23 presents that containing of being used to form that acid amides connects, azanol PEG reagent or acid amides connected contains the illustrative example through the synthetic method of the form of sheltering that contains azanol PEG reagent that connects through protection form or acid amides of azanol PEG reagent.Figure 24 and Figure 25 present that containing of being used to form that carbamate is connected, azanol PEG reagent or carbamate connected contains the illustrative example through the synthetic method of the form of sheltering that contains azanol PEG reagent that connects through protection form or carbamate of azanol PEG reagent.Figure 26 present be used to form simply contain azanol PEG reagent or simply contain azanol PEG reagent through the protection form or simply contain the illustrative example through the synthetic method of the form of sheltering of azanol PEG reagent.In addition, Figure 27 presents and has a plurality of branched illustrative example that contains azanol reagent that connects the PEG group, and further shows and a kind ofly so contain many PEG of azanol branch reagent and contain the carbonyl non-natural amino acid polypeptides and react and have the reaction that contains the oxime non-natural amino acid polypeptides that is connected many PEG branched group with formation.
Other example (for example modified PEG that can form the oxime key) that is applicable to water-soluble polymers of the present invention (for example PEG) is found in U.S. patent application case the 60/638th, No. 418; The 60/638th, No. 527 and on December 22nd, 2004 application be entitled as " Compositions containing; methods involving; and uses of non-naturalamino acids and polypeptides; " the 60/639th, No. 195, it is incorporated herein by reference in full.It is also No. the 60/696th, 210, U.S. patent application case; The 60/696th, No. 302 and on July 1st, 2005 application be entitled as " Compositions containing; methods involving; and uses of non-natural amino acids andpolypeptides; " the 60/696th, describe in 068, described reference is incorporated herein by reference in full.
Water-soluble polymers be connected to the degree of hGH polypeptide and position can regulate GH (for example, hGH) polypeptide to GH (for example, the hGH) combination of polypeptide receptor on position 1.In certain embodiments, key through so the configuration in case make GH (for example, hGH) polypeptide and GH (for example, hGH) polypeptide receptor on position 1 with about 400nM or following K d, with 150nM or following K dAnd in some cases with 100nM or following K d(K dAccording to such as people such as Spencer, J.Biol.Chem., the balance described in the 263:7862-7867 (1988) is measured in conjunction with calibrating) combination.
Be used for polymer activation and peptide bonded method and chemical reaction and describe to some extent in the literature, and be known in affiliated field.The common method that is used for polymer activation includes, but is not limited to use cyanogen bromide, periodate, glutaraldehyde, di-epoxide, Epicholorohydrin, divinylsulfone, carbodiimide, sulfuryl halide, three chlorotriazines to wait and activates functional group (referring to R.F.Taylor, (1991), PROTEIN IMMOBILISATION.FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S.S.Wong, (1992), CHEMISTRY OF PROTEINCONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. people such as T.Hermanson, (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, R.L. waits the people, Eds.POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol.469, American Chemical Society, Washington, D.C.1991).
Some summaries and monograph functionalized about PEG and keying action are available.Referring to (for example) Harris, Macromol.Chem.Phys.C25: 325-373 page or leaf (1985); Scouten, 135: the 30-65 pages or leaves (1987) of Methods in Enzymology; People such as Wong, Enzyme Microb.Technol.14: 866-874 page or leaf (1992); People such as Delgado, 9: the 249-304 pages or leaves (1992) of Critical Reviews in Therapeutic Drug Carrier Systems; Zalipsky, Bioconjugate Chem.6: 150-165 page or leaf (1995).
The method that is used for polymer activation also is found in WO 94/17039, United States Patent (USP) the 5th, 324, No. 844, WO94/18247, WO 94/04193, United States Patent (USP) the 5th, 219, No. 564, United States Patent (USP) the 5th, 122, No. 614, WO90/13540, United States Patent (USP) the 5th, 281, No. 698 and WO 93/15189, and can find the method that is used for the keying action between activated polymkeric substance and the enzyme, these enzymes include, but is not limited to blood coagulation factor VIII (WO 94/15625), oxyphorase (WO 94/09027), oxygen carrier molecule (United States Patent (USP) the 4th, 412, No. 989), rnase and superoxide-dismutase (people such as Veronese, App.Biochem.Biotech.11: 141-52 page or leaf (1985)).Whole documents and the patent quoted all are incorporated herein by reference.
Contain the non-naturally encoded amino acid GH of (such as to azido--L-phenylalanine) (for example, the hGH) PEGization of polypeptide (that is, adding any water-soluble polymers) by any facilitated method.For example, be that the mPEG derivative of alkynyl makes GH (for example, hGH) polypeptide PEGization with end.In brief, under the room temperature under stirring state with excessive solid mPEG (5000)-O-CH 2-C ≡ H is added into the GH that contains azido--L-Phe (for example, hGH) in the aqueous solution of polypeptide.Usually use and have the approaching pK of residing pH value (generally being about pH value 4 to 10) when carrying out with reaction aDamping fluid the described aqueous solution is cushioned.For example, be used for including, but is not limited to HEPES, phosphoric acid salt, borate, TRIS-HCl, EPPS and TES at the example of the suitable buffer of 7.5 times PEGization of pH value.Continue to monitor the pH value, and regulate where necessary.Usually allow this reaction to continue about 1 to 48 hour.
Make reaction product stand hydrophobic interaction chromatography subsequently and handle, (for example, hGH) polypeptide variants and free mPEG (5000)-O-CH so that PEGization GH 2-C ≡ CH and PEGization GH (for example, hGH) any high molecular weight component of polypeptide (its can when not blocking PEG and on the molecule two ends, all activated and form), (for example, hGH) polypeptide variants is molecule crosslinked thereby make GH.Condition during the hydrophobic interaction chromatography is so, so that make free mPEG (5000)-O-CH 2-C ≡ CH flows through tubing string, simultaneously any through crosslinked PEGization GH (for example, hGH) the polypeptide variants mixture (it contains one and one and above PEG group bonded GH (for example, hGH) polypeptide variants molecule) wash-out afterwards in want form.Conditions suitable will be looked cross-linked composite and change for the relative size of want binding substances, and can easily be determined by the those skilled in the art.Contain that the elutriant of wanting binding substances to some extent concentrates by ultrafiltration and by saturating filter desalination.
In case of necessity, and the PEGization GH that obtains by hydrophobic chromatography (for example, hGH) the further purifying of polypeptide by a kind of and more than one programs in the known program of those skilled in the art, these programs comprise (but being not limited to) affinity chromatography; Anionresin or cation-exchange chromatography (its use includes, but is not limited to the DEAE sepharose); The silica chromatography; Reversed-phase HPLC; Gel-filtration (use includes, but is not limited to SEPHADEX G-75); The hydrophobic interaction chromatography; Size exclusion chromatography, (size-exclusion chromatography); Metal chelate chromatography; Ultrafiltration/saturating filter; Ethanol sedimentation; Ammonium sulfate precipitation; Chromatofocusing; Displacement chromatography; Electrophoretic procedures (including, but is not limited to the isoelectrofocusing of preparation property); Difference solvability method (including, but is not limited to ammonium sulfate precipitation); Or extraction method.Apparent molecular weight can be estimated (Preneta, AZ in PROTEINPURIFICATION METHODS, A PRACTICAL APPROACH (Harris﹠amp by GPC is compared with the globular proteins standard; Angal compiles) IRL Press1989, the 293-306 page or leaf).The purity of GH (for example hGH)-PEG binding substances can be carried out mass spectroscopy then by proteolytic degradation (including, but is not limited to the trypsinase cracking) and be assessed.Pepinsky RB. waits the people, J.Pharmcol.﹠amp; Exp.Ther.297 (3): 1059-66 (2001).
Also contain the non-naturally encoded amino acid of the carbonyl GH of (such as to ethanoyl-L-phenylalanine) (for example, the hGH) PEGization of polypeptide (that is, adding any water-soluble polymers) by any facilitated method.As non-proprietary example, use amino oxygen base ethamine carbamate mPEG derivative (its molecular weight be about 0.1 to 100kDa about 1 to 100kDa or about 10 to 50kDa about 20 to 40kDa or 30kDa for example) will contain GH (hGH) the polypeptide PEGization of the non-naturally encoded amino acid of carbonyl (for example to ethanoyl-L-phenylalanine).In brief, under the room temperature under stirring state with excessive solid MPEG-oxyamine, for example mPEG (30,000)-O-CO-NH-(CH 2) 2-ONH 3 +(linear 30kDa mono methoxy-poly-(ethylene glycol)-2-amino oxygen base ethamine carbamate hydrochloride, 30K MPEG-oxyamine) is added into the GH that contains ethanoyl-L-phenylalanine (for example, hGH) in the aqueous solution of polypeptide.(for example, mol ratio hGH) can be about 2 to 15 or about 5 to 10 or about 5,6,7,8,9 or 10 to PEG:GH.Usually use damping fluid that the described aqueous solution is cushioned with the approaching pKa of residing pH value (generally being about pH value 2 to 8) when carrying out with reaction.For example, be used for including, but is not limited to by adding sodium acetate/glycine buffer that acetate is adjusted to pH4.0 at the example of the suitable buffer of 4.0 times PEGization of pH value.Usually allow this to react on and softly shake down lasting about 1 to 60 hour or about 10 to 50 hours or about 18 to 48 hours or about 39 to 50 hours under the room temperature.PEGization can be determined by sds gel.
Reaction product from (for example) free 30K MPEG-oxyamine and PEGization GH (is for example stood, hGH) any high molecular weight component of polypeptide (its can when not blocking PEG and on the molecule two ends, all activated and form) purifying, thereby (for example, hGH) polypeptide variants is molecule crosslinked to make GH.Can use any suitable purification process, for example tubing string chromatography (such as the SourceQ tubing string chromatogram of using SourceQ buffer A and SourceQ buffer B to carry out).Reaction mixture can dilute with TRIS alkali and SourceQ buffer A and MilliQ water, is loaded on the tubing string subsequently.Contain that the elutriant of wanting binding substances to some extent can further concentrate by ultrafiltration and by saturating filter desalination.
In case of necessity, the PEGization GH that obtains by chromatography (for example, hGH) the further purifying of polypeptide by a kind of and more than one programs in those skilled in the art's's known and described herein (referring to for example above) the program.Can be higher than 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 99.99% purity and obtain final PEGization GH (for example, hGH) polypeptide.Purity can be measured by known method in the affiliated field.The exemplary non-limiting method of assessment purity comprises SDS-PAGE, (for example, hGH) polypeptide, Bradford calibrating, mass spectrum (including, but is not limited to MALDI-TOF), HPLC method (such as RP HPLC, cationic exchange HPLC and gel-filtration HPLC) and known other of those skilled in the art are used for the method for profiling protein matter to use west ink dot method and ELISA calibrating to measure GH.
(for example, hGH) water-soluble polymers that is connected of the amino acid in the polypeptide can be unrestrictedly through further deriving or replacing with GH of the present invention.
The PEG derivative that contains azido-
In another embodiment of the present invention, modify GH (for example, hGH) polypeptide with the PEG derivative that contains azido-part (its will with the alkynyl partial reaction that is present on the non-naturally encoded amino acid side chain).Generally speaking, these PEG derivatives will have 1 to 100kD and be 10 to 40kDa molecular-weight average in certain embodiments.
In certain embodiments, end will have following structure for the PEG derivative of azido-:
RO-(CH 2CH 2O) n-O-(CH 2) m-N 3，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000 (that is, molecular-weight average be 5 to 40kDa).
In another embodiment, end will have following structure for the PEG derivative of azido-:
RO-(CH 2CH 2O) n-O-(CH 2) m-NH-C(O)-(CH 2) p-N 3，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2 to 10, p be 2 to 10 and n be 100 to 1,000 (that is, molecular-weight average be 5 to 40kDa).
In another embodiment of the present invention, modify GH (for example, hGH) polypeptide with the branch PEG derivative that contains terminal azido-part (each chain of wherein said PEG have 10 to 40kDa and may be the molecular weight in 5 to the 20kDa scopes).For example, in certain embodiments, end will have following array structure for the PEG derivative of azido-:
[RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)] 2CH(CH 2) m-X-(CH 2) pN 3
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2 to 10, p be 2 to 10 and n be 100 to 1,000, and X optionally is O, N, S or carbonyl (C=O), it can exist or not exist in all cases.
The PEG derivative that contains alkynyl
In another embodiment of the present invention, modify GH (for example, hGH) polypeptide with the PEG derivative that contains alkynyl part (its will with the azido-partial reaction that is present on the non-naturally encoded amino acid side chain).
In certain embodiments, end will have following array structure for the PEG derivative of alkynyl:
RO-(CH 2CH 2O) n-O-(CH 2) m-C≡CH，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be 2 to 10 and n be 100 to 1,000 (that is, molecular-weight average be 5 to 40kDa).
In another embodiment of the present invention, comprise with the PEG derivative modification that contains terminal azido-part or terminal alkynyl part (it is connected with the PEG main chain by amido linkage) and to contain the non-naturally encoded amino acid whose GH of alkynyl (for example, hGH) polypeptide.
In certain embodiments, end will have following array structure for the PEG derivative of alkynyl:
RO-(CH 2CH 2O) n-O-(CH 2)m -NH-C(O)-(CH 2) p-C≡CH，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2 to 10, p be 2 to 10 and n be 100 to 1,000.
In another embodiment of the present invention, modify to comprise with the branch PEG derivative that contains terminal alkynyl part (each chain of wherein said PEG have 10 to 40kDa and may be the molecular weight in 5 to the 20kDa scopes) and contain the amino acid whose GH of azido-(for example, hGH) polypeptide.For example, in certain embodiments, end will have following array structure for the PEG derivative of alkynyl:
[RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)] 2CH(CH 2) m-X-(CH 2) p?C≡CH，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2 to 10, p be 2 to 10 and n be 100 to 1,000, and X optionally is O, N, S or carbonyl (C=O), or do not exist.
The PEG derivative that contains the phosphine part
In another embodiment of the present invention, modify GH (for example, hGH) polypeptide with the PEG derivative that contains activated functional group (including, but is not limited to ester, carbonic ether) and further comprise aryl phosphine group (its will with the azido-partial reaction that is present on the non-naturally encoded amino acid side chain).One, these PEG derivatives will have 1 to 100kD and be 10 to 40kDa molecular-weight average in certain embodiments.
In certain embodiments, described PEG derivative will have following structure:
Figure A20058004446401721
Wherein n is 1 to 10; X can be O, N, S or does not exist, and Ph is a phenyl, and W is a water-soluble polymers.
In certain embodiments, described PEG derivative will have following structure:
Wherein X can be O, N, S or does not exist, and Ph is a phenyl, and W is a water-soluble polymers, and R can be H, alkyl, aryl, is substituted alkyl and is substituted aryl.Exemplary R group includes, but is not limited to-CH 2,-C (CH 3) 3,-OR ' ,-NR ' R " ,-SR ', halogen ,-C (O) R ' ,-CONR ' R " ,-S (O) 2R ' ,-S (O) 2NR ' R " ,-CN and-NO 2R ', R ", R  and R " " the assorted alkyl that refers to hydrogen independently of one another, is substituted or is unsubstituted, the aryl that is substituted or is unsubstituted (its include, but is not limited to replace aryl), alkyl, alkoxyl group or the thio alkoxy or the aralkyl that are substituted or are unsubstituted through 1 to 3 halogen atom.For example, when compound of the present invention comprised more than one R group, each R group was selected independently; Just as R ', R ", R  and R " " in an above group when existing, R ', R ", R  and " " group each is selected independently naturally such.As R ' and R " when being connected with same nitrogen-atoms, its therewith nitrogen-atoms combination to form 5 yuan, 6 yuan or 7 yuan of rings.For example ,-NR ' R " means and includes, but is not limited to 1-pyrrolidyl and 4-morpholinyl.Substituent argumentation is understood by those skilled in the art that term " alkyl " means and comprises the group that comprises with the carbon atom of non-hydrogen group bond, (includes, but is not limited to-CF such as haloalkyl according to above 3With-CH 2CF 3) and acyl group (include, but is not limited to-C (O) CH 3,-C (O) CF 3,-C (O) CH 2OCH 3Deng).
Other PEG derivative and general PEGization technology
Can (for example, hGH) other the exemplary PEG molecule and the PEGization method that are connected of polypeptide be included in those PEG molecules and the PEGization method described in the following patent case: No. the 2004/0001838th, U.S. Patent Publication case with GH; No. 2002/0052009; No. 2003/0162949; No. 2004/0013637; No. 2003/0228274; No. 2003/0220447; No. 2003/0158333; No. 2003/0143596; No. 2003/0114647; No. 2003/0105275; No. 2003/0105224; No. 2003/0023023; No. 2002/0156047; No. 2002/0099133; No. 2002/0086939; No. 2002/0082345; No. 2002/0072573; No. 2002/0052430; No. 2002/0040076; No. 2002/0037949; No. 2002/0002250; No. 2001/0056171; No. 2001/0044526; No. 2001/0021763; United States Patent (USP) the 6th, 646, No. 110; The 5th, 824, No. 778; The 5th, 476, No. 653; The 5th, 219, No. 564; The 5th, 629, No. 384; The 5th, 736, No. 625; The 4th, 902, No. 502; The 5th, 281, No. 698; The 5th, 122, No. 614; The 5th, 473, No. 034; The 5th, 516, No. 673; The 5th, 382, No. 657; The 6th, 552, No. 167; The 6th, 610, No. 281; The 6th, 515, No. 100; The 6th, 461, No. 603; The 6th, 436, No. 386; The 6th, 214, No. 966; The 5th, 990, No. 237; The 5th, 900, No. 461; The 5th, 739, No. 208; The 5th, 672, No. 662; The 5th, 446, No. 090; The 5th, 808, No. 096; The 5th, 612, No. 460; The 5th, 324, No. 844; The 5th, 252, No. 714; The 6th, 420, No. 339; The 6th, 201, No. 072; The 6th, 451, No. 346; The 6th, 306, No. 821; The 5th, 559, No. 213; The 5th, 747, No. 646; The 5th, 834, No. 594; The 5th, 849, No. 860; The 5th, 980, No. 948; The 6th, 004, No. 573; The 6th, 129, No. 912; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, 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 96/07670, EP 605 963, EP 510 356, EP400 472, EP 183 503 and EP 154 316, it is incorporated herein by reference.Described herein any PEG molecule can use in any form, includes, but is not limited to strand, side chain, multi-arm chain, simple function, multi-functional or its any combination.
Enhancing is to sero-abluminous avidity
Also can (for example, hGH) polypeptide merges, to regulate GH (for example, the hGH) transformation period of polypeptide in serum with multiple molecule and GH of the present invention.In certain embodiments, (for example, hGH) polypeptide is connected or merges, to strengthen for the sero-abluminous affinity of the endogenous of animal with molecule and GH of the present invention.
For example, in some cases, (for example, hGH) reorganization of polypeptide and albumin bound sequence is merged to carry out GH.Exemplary albumin bound sequence includes, but is not limited to albumin bound territory from streptococcus protein G (referring to people such as for example Makrides, people such as J.Pharmacol.Exp.Ther.277:534-542 (1996) and Sjolander, J, Immunol.Methods 201:115-123 (1997)), or albumin binding peptide (such as at people such as for example Dennis, J.Biol.Chem.277: described in the 35035-35043 page or leaf (2002)).
In other embodiments, use lipid acid with GH of the present invention (for example, hGH) polypeptide acidylate.In certain embodiments, lipid acid promotes to combine with sero-abluminous.Referring to people such as for example Kurtzhals, Biochem.J.312: 725-731 page or leaf (1995).
In other embodiments, (for example, hGH) polypeptide is directly to merge with serum albumin (including, but is not limited to the human serum albumin) to GH of the present invention.Those skilled in the art will realize that and also can make multiple other molecule and GH of the present invention (for example, hGH) polypeptide is connected, with combining of adjusting and serum albumin or other serum component.
X.GH (for example, the hGH) glycosylation of polypeptide
The present invention includes and wherein incorporate one or more non-naturally encoded amino acid whose GH (for example, hGH) polypeptide into carbohydrate residue.These carbohydrate residues can be natural (including, but is not limited to N-acetylamino glucose) or non-natural (including, but is not limited to 3-fluorine semi-lactosi).These carbohydrates can be connected with non-naturally encoded amino acid by N connects or O connects glycosidic link (including, but is not limited to N-ethanoyl semi-lactosi-L-Serine) or non-natural key (including, but is not limited to the glucosides that oxime or corresponding C connect or S connects).
Carbohydrate (including, but is not limited to glycosyl) part can be added in vivo or sv GH (for example, hGH) polypeptide.In some embodiments of the invention, use to modify to comprise and contain the non-naturally encoded amino acid whose GH of carbonyl (for example, hGH) polypeptide is to produce the corresponding glycosylated polypeptides that is connected by the oxime key with amino oxygen base deutero-carbohydrate.In case after being connected to non-naturally encoded amino acid, just can be further refining by handling carbohydrate with glycosyltransferase and other enzyme, to produce (for example, hGH) polypeptide bonded oligose with GH.Referring to people J.Am.Chem.Soc.125 such as for example H.Liu: 1702-1703 page or leaf (2003).
In some embodiments of the invention, use the glycan for preparing, has definite structure as the amino oxygen radical derivative directly to modify to comprise and contain the non-naturally encoded amino acid whose GH of carbonyl (for example, hGH) polypeptide.Those skilled in the art will realize that and to use other functional group (comprising azido-, alkynyl, hydrazides, hydrazine and Urea,amino-) that carbohydrate is connected to non-naturally encoded amino acid.
In some embodiments of the invention, then can modify with Hu Shi root [3+2] cycloaddition reaction of (including, but is not limited to) alkynyl or azido-derivative respectively and comprise azido-or contain the non-naturally encoded amino acid whose GH of alkynyl (for example, hGH) polypeptide by (including, but is not limited to).This method allows to come modifying protein with high selectivity.
XI.GH supergene family member's dimer and polymer
The present invention also provides GH supergene family member's combination (to include, but is not limited to GH, for example hGH and hGH analogue), such as homodimer, heterodimer, homology polymer or heteropolymer (promptly, the tripolymer or the tetramer etc.), wherein contain one or more non-naturally encoded amino acid whose GH supergene family member polypeptide (such as GH, hGH for example) combines with any other polypeptide of another GH supergene family member or its varient or non-GH supergene family member or its varient, directly or by connexon be connected to the polypeptide main chain.Because its molecular weight is higher compared to monomer, therefore GH supergene family member is (such as GH, hGH for example) dimer or polymer binding substances can represent new or desired characteristic, its include, but is not limited to different pharmacological properties, PK (pharmacokinetic) profile, drug effect characteristic, with respect to monomer GH supergene family member through regulating the transformation period or through regulating plasma half-life.In certain embodiments, GH supergene family member of the present invention (such as GH, for example hGH) dimer will be regulated the dimerisation of GH supergene family member acceptor.In other embodiments, GH supergene family member's dimer of the present invention or polymer will serve as GH supergene family member receptor antagonist, agonist or modulator.
In certain embodiments, be present in one or more GH (for example hGH) molecule that contains among dimer or the polymeric GH (for example hGH) and comprise the non-naturally encoded amino acid that the water-soluble polymers interior with being present in position II calmodulin binding domain CaM is connected.Thereby dimer or polymeric each molecule are easy to (for example, hGH) the polypeptide receptor combination, but can not pass through II interface, position and the 2nd GH (for example, hGH) polypeptide receptor combination by I interface, position and GH.Therefore, GH (for example, hGH) polypeptide dimer or polymer can with two different GH (for example, hGH) the position I combining site combination of each acceptor in the polypeptide receptor, but because GH (for example, hGH) peptide molecule has and is connected to the amino acid whose water-soluble polymers of non-genetic coding that is present in the II zone, position, therefore GH (for example, hGH) polypeptide receptor can't with GH (for example, hGH) the position II of polypeptide ligand zone combination, and dimer or polymer serve as GH (for example, hGH) polypeptide antagonist.In certain embodiments, be present in one or more GH (for example hGH) molecule that contains among dimer or the polymeric GH (for example hGH) and comprise the non-naturally encoded amino acid that the water-soluble polymers interior with being present in position I calmodulin binding domain CaM is connected, combine with position II zone with permission.Perhaps, in certain embodiments, be present in one or more GH (for example hGH) molecule that contains among dimer or the polymeric GH (for example hGH) and comprise the non-naturally encoded amino acid that is connected with the water-soluble polymers that is not present in position I or the position II calmodulin binding domain CaM, so as to make two zones all can in conjunction with.In certain embodiments, use have position I can with, position II can with or both equal available GH (for example hGH) molecular combinations.GH (for example hGH) molecular combinations (wherein at least one molecule has the bonded of can be used for position I, and at least one molecule have can be used for bonded position II) can provide have the molecule of the activity of wanting or characteristic.In addition, have GH (for example hGH) molecular combinations that can be used for bonded position I and position II simultaneously and can produce super short effect GH (for example hGH) molecule.
In certain embodiments, GH supergene family member polypeptide (including, but is not limited to) directly connects by Asn-Lys amine key or Cys-Cys disulphide.In certain embodiments, GH supergene family member's polypeptide through connecting and/or the non-GH supergene family member through connecting will comprise different non-naturally encoded amino acid so that dimerisation, its include, but is not limited to a GH (for example, hGH) alkynyl in polypeptide non-naturally encoded amino acid with will be at the azido-in second of the 2nd GH supergene family member polypeptide non-naturally encoded amino acid by Hu Shi root [3+2] cycloaddition in conjunction with being connected.Perhaps, the one GH supergene family member and/or the non-GH supergene family member through connecting, comprise the non-naturally encoded amino acid whose polypeptide of ketone group containing and can contain non-naturally encoded amino acid whose the 2nd GH supergene family member polypeptide of azanol in conjunction with being connected to comprise, these polypeptide react by forming corresponding oxime.
Perhaps, two GH supergene family member's polypeptide and/or the non-GH supergene family member through connecting connect by connexon.Any Heterobifunctional be can use or two GH supergene family members and/or non-GH supergene family member, polypeptide (it can have identical or different primary sequence) connected through connecting with the difunctionality connexon.In certain embodiments, be used for can be difunctionality PEG reagent with GH supergene family member and/or through the non-GH supergene family member of connection, the connexon that polypeptide chain is connected together.This connexon can have the molecular weight or the molecular length of broad range.Larger molecular weight or than the connexon of small molecular weight be used in GH supergene family member be connected between the entity GH supergene family member and GH supergene family member acceptor between or be connected between entity and the GH supergene family member acceptor spatial relation of wanting or structure be provided.Have longer molecular length or than the connexon of short molecule length also be used in GH supergene family member be connected between the entity GH supergene family member and its acceptor between or be connected between entity and the GH supergene family member acceptor space of wanting and flexible be provided.Similarly, the connexon with special shape or structure is used in GH supergene family member and arrives before or after the target GH supergene family member or connect entity and give special shape or structure.Thisly can be molecule for GH supergene family member with the optimization that is connected the spatial relation between the entity new, that connect adjusting or desired characteristic is provided.
In certain embodiments, the invention provides water-soluble difunctionality connexon, it has the dumbbell structure, and this structure comprises: a) be positioned at azido-, alkynyl, hydrazine, hydrazides, the azanol at least the first end of main polymer chain or contain carbonyl moiety; And b) is positioned at least one second functional group on main polymer chain second end.This second functional group can be identical or different with first functional group.In certain embodiments, second functional group not with first functional group reactions.In certain embodiments, the invention provides the water-soluble cpds of at least one arm that comprises the branch molecular structure.For example, this branch molecular structure can be dendritic.
In certain embodiments, the invention provides the polymer that comprises one or more GH supergene family members (such as GH, for example hGH), it is by forming with the water-soluble activated polymer reaction with following structure:
R-(CH 2CH 2O) n-O-(CH 2) m-X，
Wherein n is 5 to 3,000, and m is 2 to 10, and X can be azido-, alkynyl, hydrazine, hydrazides, amino oxygen base, azanol, ethanoyl or contains carbonyl moiety, and R is capping group, functional group or leavings group, and it can be identical or different with X.R can be the functional group that (for example) is selected from the group that is made up of following each group: hydroxyl; through the protection hydroxyl; alkoxyl group; N-maloyl imines ester; 1-benzotriazole base ester group; N-maloyl imido grpup carbonic ether; 1-benzotriazole base carbonic ether; acetal; aldehyde; aldehydrol; thiazolinyl; acrylate; methacrylic ester; acrylamide; active sulfone; amine; the amino oxygen base; through protection amine; hydrazides; through the protection hydrazides; through protection mercaptan; carboxylic acid; through the protection carboxylic acid; isocyanic ester; lsothiocyanates; maleimide; vinyl sulfone(Remzaol; two thiopyridines; vinyl pyridine; iodo-acid amide; epoxide; oxalic dialdehyde; diketone; methanesulfonates; tosylate; the trifluoro esilate; alkene and ketone.
XII.hGH polypeptide active and hGH polypeptide are for the measurement of the affinity of hGH polypeptide receptor
[586] can be according to people such as McFarland, Science, 245: the 494-499 pages or leaves (1989) and Leung, people such as D., Nature, preparation hGH acceptor described in 330: the 537-543 page or leaf (1987).The hGH polypeptide active can use standard or in vivo known or in vitro calibrating mensuration.For example, exist under the situation proliferating cells system (for example expressing the clone of hGH acceptor or lactagogue acceptor) to can be used for monitoring the hGH receptors bind at hGH.Referring to for example Clark, people such as R., J.Biol.Chem.271 (36): the 21969th page (1996); People such as Wada, Mol.Endocrinol.12: 146-156 page or leaf (1998); Gout, people Cancer Res.40 such as P.W., 2433-2436 page or leaf (1980); WO 99/03887.For non-PEGization that comprises alpha-non-natural amino acid or PEGization hGH polypeptide, hormone can be by using BIAcore for the affinity of its acceptor TMBiosensor (Pharmacia) is measured.Referring to for example No. the 5th, 849,535, United States Patent (USP), Spencer, people such as S.A., J.Biol.Chem., 263: the 7862-7867 pages or leaves (1988).Be used to test the active in vivo animal model of hGH and comprise that those are at people such as (for example) Clark, J.Biol.Chem.271 (36): the model described in the 21969-21977 page or leaf (1996).Can be according to Cunningham, people such as B., Science, 254: the 821-825 pages or leaves (1991) and Fuh, G. wait the people, Science carries out the calibrating for the dimerization ability that comprises one or more non-naturally encoded amino acid whose hGH polypeptide at 256: the described in 1677-1680 page or leaf (1992).The document and the patent of all references are incorporated herein by reference.
Document editing about calibration method is not exhaustive, and the those skilled in the art is applicable to approval other calibrating of the test net result of wanting.
XIII. for the measurement of usefulness, functional in vivo transformation period and pharmacokinetic parameter
An importance of the present invention is the biological half time by the prolongation that structure obtained of hGH polypeptide (under the bonded situation that has or do not have polypeptide and water-soluble polymers part).The rapid reduction of hGH polypeptide serum-concentration has made that assessment has importance for the biological respinse of the treatment of using combination or non-binding hGH polypeptide and its varient.After subcutaneous or intravenous injection dispensing, combination of the present invention or non-binding hGH polypeptide and its varient also can have lasting serum half-life, and feasible the measurement surely by (for example) ELISA method or by the primary screen Selected Inspection becomes possibility.Can use available from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, ELISA TX) or RIA cover group.Carry out the in vivo measurement of biological half-life as described herein.
The usefulness and the functional in vivo transformation period that comprise non-naturally encoded amino acid whose hGH polypeptide can be according to Clark, people such as R., and J.Biol.Chem.271 (36): the method described in the 21969-21977 page or leaf (1996) is measured.
The pharmacokinetic parameter that comprises non-naturally encoded amino acid whose hGH polypeptide can be assessed in common Sprague-Dawley male rat (N=5 animal/treatment group).Animal will be accepted in the 25 μ g/ rat veins or the single dose of 50 μ g/ subcutaneous rat, according to predetermined time-histories (generally contain for comprise non-naturally encoded amino acid, not with water-soluble polymers bonded GH (for example, hGH) polypeptide is about 6 hours, and for comprising non-naturally encoded amino acid and gathering about 5 to 7 parts of blood samples with water-soluble polymers bonded GH (for example, hGH) polypeptide about 4 days).In some materials, fully studied GH (for example, the hGH) pharmacokinetic data of polypeptide, and can be with it with (for example, hGH) data that polypeptide obtained directly compare for comprising non-naturally encoded amino acid whose GH.About (for example, hGH) the relevant research of polypeptide is referring to people such as Mordenti J., Pharm.Res.8 (11): 1351-59 page or leaf (1991) with GH.
Pharmacokinetic parameter also can be assessed in primate (for example macaque).Usually, throw in subcutaneous or intravenous mode and to give single injection, and monitor Serum GH (for example hGH) content in time.About further describing, referring to for example " example ".
In certain embodiments, the invention provides throwing and have when giving Mammals at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16 or the GH (for example hGH) of average serum transformation period more than 16 hours in subcutaneous mode.In certain embodiments, the invention provides throwing and have when giving Mammals at least about 0.25,0.5,0.75,1,2,3,4,5,6,7,8,9,10,11,12 or the GH (for example hGH) of average serum transformation period more than 12 hours in subcutaneous mode." on average " serum half-life is the mean value of at least three animals or at least four animals or at least five animals or five above animals.In certain embodiments, Mammals is a rat; In certain embodiments, Mammals is a primate, such as macaque, or such as the mankind.In certain embodiments, the invention provides PEGization GH (for example PEGization hGH), it has average serum transformation period of being equivalent to non-PEGization form GH (for example hGH) at least about 2 times, 3 times, 4 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, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, average serum transformation period more than 50 times or 50 times throwing in subcutaneous mode when giving.In certain embodiments, the invention provides PEGization GH (for example PEGization hGH), it has average serum transformation period of being equivalent to non-PEGization form GH (for example hGH) at least about 2 times, 3 times, 4 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, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, average serum transformation period more than 50 times or 50 times throwing in the intravenously mode when giving." on average " serum half-life is the mean value of at least three animals or at least four animals or at least five animals or five above animals.In certain embodiments, Mammals is a rat; In certain embodiments, Mammals is a primate, such as macaque, or such as the mankind.In certain embodiments, GH is GH (for example hGH).In certain embodiments, tethelin is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.GH can be GH (for example hGH).In certain embodiments, GH (for example hGH) comprises non-naturally encoded amino acid, such as containing the non-naturally encoded amino acid of carbonyl.In certain embodiments, non-naturally encoded amino acid is ketone group containing amino acid, for example to acetylphenylalanine.In certain embodiments, GH (for example hGH) contains non-naturally encoded amino acid, for example in GH (for example hGH), replace on the position corresponding to the amino acid 35 among the SEQ ID NO:2 to acetylphenylalanine.Water-soluble polymers can be PEG.Suitable PEG comprises linear PEG and branch PEG; Can use described any PEG herein.In a particular embodiment, PEG is about 0.1 to 100kDa or about 1 to 100kDa or about 10 to 50kDa or about 20 to 40kDa or the linear PEG of about 30kDa.In certain embodiments, medical composition contains the GH (for example hGH) that is connected with the PEG of 30kDa by the oxime key, wherein said oxime key corresponding to the amino acid 35 among the SEQ ID NO:2 locational to acetylphenylalanine and PEG between.
(for example, hGH) given activity of polypeptide can be measured by known multiple calibrating in the affiliated field according to GH of the present invention.Obtain according to the present invention and the GH of purifying (for example, hGH) polypeptide mutein or its segmental biological activity can be by method or the known method of those skilled in the art described or that quote are tested herein.
XIV. offer medicine and medical composition
Polypeptide of the present invention or protein (include, but is not limited to GH (for example hGH), synthetic enzyme, comprise one or more amino acid whose protein etc.) optionally (include, but is not limited to) be used for the treatment of purposes with suitable medical supporting agent combination.For example, described composition comprises the compound for the treatment of significant quantity and pharmaceutically acceptable supporting agent or vehicle.Described supporting agent or vehicle include, but is not limited to salt solution, through buffer saline, glucose, water, glycerine, ethanol and/or its combination.Make prescription be suitable for dispensing.Generally speaking, throw give method of protein for the those skilled in the art for known, and can be applicable to throw and give polypeptide of the present invention.
In certain embodiments, the invention provides and contain the hormonal composition medical composition of (it comprises the tethelin that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key); And pharmaceutically acceptable vehicle.GH can be hGH.In certain embodiments, GH (for example hGH) comprises non-naturally encoded amino acid, such as containing the non-naturally encoded amino acid of carbonyl.In certain embodiments, non-naturally encoded amino acid is ketone group containing amino acid, for example to acetylphenylalanine.In certain embodiments, GH (for example hGH) contains non-naturally encoded amino acid, for example in GH (for example hGH), replace on the position corresponding to the amino acid 35 among the SEQ ID NO:2 to acetylphenylalanine.Water-soluble polymers can be PEG.Suitable PEG comprises linear PEG and branch PEG; Can use described any PEG herein.In a particular embodiment, PEG is about 0.1 to 100kDa or about 1 to 100kDa or about 10 to 50kDa or about 20 to 40kDa or the linear PEG of about 30kDa.In certain embodiments, medical composition contains the GH (for example hGH) that is connected with the PEG of 30kDa by the oxime key, wherein said oxime key corresponding to the amino acid 35 among the SEQ ID NO:2 locational to acetylphenylalanine and PEG between.
According to the known method of those skilled in the art, if necessary in one or more suitable in vivo and/the test therapeutic composition that comprises one or more polypeptide of the present invention in the animal disease model in vitro, to confirm usefulness, tissue metabolism and estimation dosage.Specifically, can by alpha-non-natural amino acid herein with respect to the natural amino acid homologue (include, but is not limited to modified with the GH that comprises one or more alpha-non-natural amino acids (for example, hGH) polypeptide and natural amino acid GH are (for example, hGH) comparison of polypeptide), promptly activity, stability or other suitable the measuring in relevant calibrating tentatively determined dosage.
The molecule introducing is offerd medicine with blood or the histiocytic final any approach that contacts by being generally used for.Give non-natural amino acid polypeptides of the present invention with any suitable method throwing, optionally use one or more pharmaceutically acceptable supporting agents.Throwing the appropriate method give described polypeptide in literary composition of the present invention is available, although and can use more than one approach to throw and give particular composition, particular approach can provide usually compared to more direct and more effective effect of other approach or reaction.
Pharmaceutically acceptable supporting agent is to a certain extent by the throw particular composition that gives and be used for throwing and give described method for compositions and determine.Therefore, there is multiple suitable prescription in medical composition of the present invention.
HGH polypeptide of the present invention (including, but is not limited to PEGization hGH) can be thrown by any conventional route that is applicable to protein and polypeptide and give, these approach include, but is not limited to non-through the intestines mode, and for example injection or infusion (include, but is not limited to the injection or the infusion of subcutaneous or intravenously or any other form.Peptide composition can be thrown by some approach and give, these approach include, but is not limited to through the oral cavity, intravenously, intraperitoneal, intramuscular, through skin, subcutaneous, local, hypogloeeis or rectal.The composition (modified or not modified) that comprises non-natural amino acid polypeptides also can give via the liposome throwing.Described dosing way and proper formulation are generally known for the those skilled in the art.Comprising non-naturally encoded amino acid whose hGH polypeptide (including, but is not limited to PEGization hGH) can use separately or be used in combination with other suitable ingredients (such as medical supporting agent).
Also can (for example, hGH) polypeptide combines individually or with other suitable ingredients and is formed on (being that it can be " spray form ") in the aerosol formulations, to give via sucking to throw with the GH that comprises alpha-non-natural amino acid.Aerosol formulations can be placed the propelling agent accepted of pressurization, such as Refrigerant 12, propane, nitrogen etc.
Be applicable to that non-prescription through intestines dispensing (such as by intraarticular, intravenously, intramuscular, intracutaneous, intraperitoneal and subcutaneous route) comprises water-based and the aseptic injectable solution of opening such as nonaqueous, it can contain antioxidant, buffer reagent, fungistat and make this prescription and expection recipient's the solute of blood etc.; And water-based and nonaqueous sterile suspensions, it can comprise suspension agent, solubilizing agent, thickening material, stablizer and sanitas.The prescription of hGH can be present in unitary dose or the multiple doses sealed vessel (such as ampoule and phial).
Non-is preferred medication administration method through intestines dispensing and intravenously dispensing.Specifically, the dosing way that has been used for natural amino acid homologue treatment (include, but is not limited to those and be used for EPO, GH, G-CSF, GM-CSF, IFN, interleukin, antibody and/or any protein that other transmits in medical mode), together with currently used prescription, provide the screening formulation of preferred dosing way and polypeptide of the present invention.
According to application, throwing the dosage that gives the patient in literary composition of the present invention is enough to have in time useful therapeutic response or other suitable active in patient's body.Determine dosage by activity, stability or the serum half-life of the usefulness of specific support or prescription, used non-natural amino acid polypeptides, patient's illness and patient's to be treated body weight and surface-area.Also by following existence, the nature and extent of in particular patient, throwing any adverse side effect that gives specific support, prescription etc. to determine the dosage size.
When in treatment that is determined at disease (including, but is not limited to cancer, heredopathia, diabetes, AIDS or similar disease) or prevention, waiting to throw the significant quantity of the carrier that gives or prescription, the process of doctor's assessments blood plasma level, prescription toxicity, disease and/or produce anti-non-natural amino acid polypeptides antibody wherein.
For the patient of (for example) 70 kilograms throw the dosage that gives usually with scope that the currently used proteic dosage of regulating at the change activity or the serum half-life of correspondent composition of treatment equates in.Carrier of the present invention or prescription can replenish the treatment situation by any known conventional treatment (comprising that antibody is given in throwing, throwing is given vaccine, thrown and give cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleotide homology thing, biological response modifier etc.).
For dispensing, according to the LD-50 by corresponding prescription or ED-50 and/or to (including, but is not limited to) as corresponding to the patient quality and holistic health when using under the various concentration the determined ratio of observation of any side effect of alpha-non-natural amino acid throw and give prescription of the present invention.Can finish dispensing by single dose or fractionated dose.
The patient of infusion suffers from fever, catches cold or myalgia if stand to fill a prescription, and he can take acetylsalicylic acid (aspirin), Ibuprofen BP/EP (ibuprofen), acetaminophen (acetaminophen) or other antipyretic and analgesic of suitable dosage so.Premedicated before 30 minutes continuing infusion acetylsalicylic acid (aspirin), acetaminophen (acetaminophen) or (including, but is not limited to) diphenhydramine (diphenhydramine) for the respond patient of (such as fever, myalgia or catch cold) of infusion.Pethidine (Meperidine) is to be used for making febrifuge and antihistaminic agent that quick response is more serious to catch cold and myalgia.Cell infusion is slowed down or stopped to severity according to reaction.
Human GH polypeptide of the present invention can directly be thrown and give the Mammals person under inspection.By any being usually used in hGH polypeptide introducing person under inspection's approach being offerd medicine.According to the hGH peptide composition of the embodiment of the invention comprise those be applicable to (including, but is not limited to the hypogloeeis), vagina in oral cavity, rectum, part, suction (including, but is not limited to), the cheek via aerosol, non-(be skin and mucomembranous surface through intestines (include, but is not limited in subcutaneous, intramuscular, intracutaneous, intraarticular, the pleura, in the intraperitoneal, brain, intra-arterial or intravenously), part, comprise the tracheae surface) and through the composition of skin dispensing, yet the optimum approach under any given situation will be decided on sanatory character of institute and severity.Dispensing can be partial or whole body.The prescription of compound can be present in single dose or the multiple doses sealed vessel (such as ampoule and phial).(for example, hGH) polypeptide prepares in mixture with unitary dose injectable forms (including, but is not limited to solution, suspension or emulsion) and pharmaceutically acceptable supporting agent GH of the present invention.(for example, hGH) polypeptide also can give by continuous infusion (use includes, but is not limited to minipump (such as osmotic pump)), independent bolus or the throwing of sustained-release storage type prescription GH of the present invention.
The prescription that is suitable for offeing medicine comprise water-based and non-aqueous solution, etc. open sterile solution, it can contain makes this prescription wait antioxidant, buffer reagent, fungistat and the solute of opening; And water-based and nonaqueous sterile suspensions, it can comprise suspension agent, solubilizing agent, thickening material, stablizer and sanitas.Solution and suspension can be by sterilized powder, particle and the tablet preparation of previous described kind.
Lyophilize is a kind of technology that is usually used in marking protein, and it is used for removing moisture from described protein preparation.Lyophilize or lyophilization are a kind of with the at first freezing method of following by distillation will be iced or chilled solvent removes in vacuum environment of material to be dried.In the prescription of freeze-drying in advance, may comprise vehicle, with stability during the enhancing freezing dry process and/or the stability of improvement lyophilized products when storing.Pikal, people Pharm.Res.8 (3) such as M.Biopharm.3 (9) 26-30 pages or leaves (1990) and Arakawa: 285-291 page or leaf (1991).
The spraying drying of medicine also is known for the those skilled in the art.For example, referring to Broadhead, people such as J. are at Drug Dev.hid.Pharm, 18 (11﹠amp; 12), " the The Spray Dryingof Pharmaceuticals, " in the 1169-1206 page or leaf (1992).Except small-molecule drug, with multiple biomaterial spraying drying, and these materials comprise: enzyme, serum, blood plasma, microorganism and yeast.Spraying drying is a kind of suitable technology, because it can make the liquid medicine preparation be converted into powder meticulous, dustless or cohesion in a one-step process.Basic fundamental comprises following four steps: a) will present the material solution atomization and be spraying; B) spraying-air contact; C) spraying drying; D) the drying product is separated with dry air.United States Patent (USP) the 6th, 235, No. 710 and the 6th, 001, No. 800 (it is incorporated herein by reference) described and prepared recombinant erythropoietin by spraying drying.
Medical composition of the present invention can comprise pharmaceutically acceptable supporting agent, vehicle or stablizer.Pharmaceutically acceptable supporting agent is to a certain extent by the throw particular composition that gives and be used to throw the ad hoc approach that gives described composition and determine.Therefore, there is multiple suitable prescription (comprising optionally pharmaceutically acceptable supporting agent, vehicle or stablizer) (referring to for example Remington ' s Pharmaceutical Sciences, the 17th version, 1985) in medical composition of the present invention).
Suitable supporting agent comprises buffer reagent, and it contains Succinic Acid, phosphoric acid, boric acid, HEPES, citric acid, Histidine or histidine derivative, imidazoles, acetate, heavy carbonic and other organic acid; Antioxidant, it includes, but is not limited to xitix; Low molecular weight polypeptide, it includes, but is not limited to those polypeptide less than about 10 residues; Protein, it includes, but is not limited to serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, it includes, but is not limited to polyvinylpyrrolidone; Amino acid, it includes, but is not limited to glycine, glutamine, asparagine, arginine, Histidine or histidine derivative, methionine(Met), L-glutamic acid or Methionin; Monose, disaccharides and other carbohydrate, it includes, but is not limited to trehalose, sucrose, glucose, seminose or dextrin; Sequestrant, it includes, but is not limited to EDTA; Divalent-metal ion, it includes, but is not limited to zine ion, cobalt ion or cupric ion; Sugar alcohol, it includes, but is not limited to N.F,USP MANNITOL or sorbyl alcohol; The salify gegenion, it includes, but is not limited to Tween TM(include, but is not limited to Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20), Pluronics TMWith other pfpe acid, it includes, but is not limited to pfpe acid F68 (poloxamer (poloxamer) 188) or PEG.Suitable surfactant comprise (such as but not limited to) based on poly-(ethylene oxide)-poly-(propylene oxide)-poly (ethylene oxide) (i.e. (PEO-PPO-PEO)) or poly-(propylene oxide)-poly-(ethylene oxide)-the gather polyethers of (propylene oxide) (i.e. (PPO-PEO-PPO)) or its combination.PEO-PPO-PEO and PPO-PEO-PPO can trade mark Pluronics TM, R-Pluronics TM, Tetronics TMAnd R-Tetronics TM(BASF Wyandotte Corp., Wyandotte Mich.) buys, and further at United States Patent (USP) the 4th, 820, describes in No. 352 (it is incorporated herein by reference in full).Other ethene/polyethylene block polymkeric substance can be suitable tensio-active agent.The combination of tensio-active agent or tensio-active agent can be used for making PEGization hGH stable at one or more pressure (its include, but is not limited to caused by stirring pressure).Above-mentioned some reagent can be described as " swelling agent ".Some also can be described as " tonus modifier ".
GH of the present invention (for example, hGH) also can throw by sustained release system or as the part of sustained release system and give by polypeptide (it comprises the GH that those are connected with water-soluble polymers such as PEG).Sustained-release composition includes, but is not limited to the semipermeable polymers matrix of formed article form (it includes, but is not limited to film or microcapsule).Lasting release matrix comprises biocompatible material, such as poly-(methacrylic acid 2-hydroxyethyl ester) (people such as Langer, J.Biomed.Mater.Res., 15: the 267-277 pages or leaves (1981); Langer, Chem.Tech., 12: the 98-105 pages or leaves (1982)), ethyl vinyl acetate people such as (, see above) Langer or poly--D-(-)-3-hydroxybutyric acid (EP 133,988), polylactide (poly(lactic acid)) (United States Patent (USP) the 3rd, 773, No. 919; EP 58,481), poly-glycollide (polymkeric substance of oxyacetic acid), polylactide-co-glycolide multipolymer (multipolymer of lactic acid and oxyacetic acid) polyanhydride, the multipolymer of L-L-glutamic acid and γ-ethyl-L-glutamate (people such as Sidman, Biopolymers, 22, the 547-556 pages or leaves (1983)), poly-(ortho acid) ester, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, lipid acid, phosphatide, polysaccharide, nucleic acid, polyamino acid, amino acid is (such as phenylalanine, tyrosine, Isoleucine), polynucleotide, polyethylene propylene, polyvinylpyrrolidone and silicone.Sustained-release composition also comprises the compound with liposome bag quilt.The liposome that contains this compound by following self known method preparation: DE 3,218, and 121; People such as Eppstein, Proc.Natl.Acad.Sci.U.S.A., 82: the 3688-3692 pages or leaves (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 77: the 4030-4034 pages or leaves (1980); EP 52,322; EP 36,676; United States Patent (USP) the 4th, 619, No. 794; EP 143,949; United States Patent (USP) the 5th, 021,234; No. 8008, Japanese patent application case 83-11; United States Patent (USP) the 4th, 485, No. 045 and the 4th, 544, No. 545; And EP 102,324.The document and the patent of all references are incorporated herein by reference.
(for example, hGH) polypeptide: DE 3,218,121 can to prepare GH with liposome bag quilt by for example method described in following document and the patent; People such as Eppstein, Proc.Natl.Acad.Sci.U.S.A., 82: the 3688-3692 pages or leaves (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 77: the 4030-4034 pages or leaves (1980); EP 52,322; EP 36,676; United States Patent (USP) the 4th, 619, No. 794; EP 143,949; United States Patent (USP) the 5th, 021, No. 234; Japanese patent application case 83-118008 number; United States Patent (USP) the 4th, 485, No. 045 and the 4th, 544, No. 545; And EP 102,324.The composition of liposome and size are known or can easily be measured with experiment method by the those skilled in the art.Some examples of liposome are for example being described in following document and the patent: people such as Park JW, 92: the 1327-1331 pages or leaves (1995) of Proc.Natl.Acad.Sci.USA; Lasic D and Papahadjopoulos D (eds): MEDICALAPPLICATIONS OF LIPOSOMES (1998); People such as Drummond DC are at Teicher B (ed): C ANCERD RUGD ISCOVERY ANDD EVELOPMENT(2002) the Liposomal drug delivery systems forcancer therapy in; People such as Park JW, Clin.Cancer Res.8: 1172-1181 page or leaf (2002); People such as Nielsen UB, Biochim.Biophys.Acta 1591 (1-3): 109-118 page or leaf (2002); People such as Mamot C, Cancer Res.63: 3154-3161 page or leaf (2003).The document and the patent of all references are incorporated herein by reference.
In literary composition of the present invention, throw the dosage that gives the patient and should be enough in patient's body, cause in time useful reaction.Generally speaking, the non-GH of the present invention that gives through the intestines throwing (for example in every dose, hGH) polypeptide pharmaceutically effectively total amount in weight in patients about 0.01 μ g/kg/ day to about 100 μ g/kg or about 0.05mg/kg extremely in the scope of about 1mg/kg, yet this should decide on therapy.Administration frequency also should be decided on therapy, and compared to be applicable to through approval human commercially available GH (for example, hGH) polypeptide products, its frequency may be higher or lower.Generally speaking, can throw by above-mentioned any dosing way and give PEGization GH of the present invention (for example, hGH) polypeptide.Some embodiment this, the invention provides a kind of composition, it is described in this article for comprising described any GH (for example, hGH) polypeptide in storage and the sufficiently stable medical composition of administration instructions about how to take medicine herein.The method of stable testing is known in affiliated field.
XV. GH of the present invention (for example, the hGH) therepic use of polypeptide
(for example, hGH) polypeptide is applicable to the various illnesss of treatment to GH of the present invention.
(for example, hGH) the agonist polypeptide is treated growth defect, immune disorder applicable to (for example) to GH of the present invention, and is applicable to the promotion heart function.The individuality of suffering from growth defect comprises that (for example) suffer from the Turner's synodrome (individuality of Turner ' sSyndrome), GH defective individuality (comprising children), about 2 to 3 years its normal growth curves of experience children's (being also referred to as " short and small normal child " sometimes) of poor growth or delay occur and have wherein used chemical mode (promptly passing through glucocorticoid treatment) or blocked the individuality of rhIGF-1 (IGF-I) to the reaction of GH by natural condition (in the adult patients that the reaction of GH is reduced naturally such as IGF-I therein) before the growth plate closure.HGH polypeptide of the present invention suffers from the individuality of following illness applicable to treatment: the paediatrics growth hormone deficiency, special send out property of short and small stature, the Childhood outbreak grownup's growth hormone deficiency, the grownup's growth hormone deficiency or the Secondary cases growth hormone deficiency of Adulthood outbreak.The grownup who suffers from the Adulthood growth hormone deficiency after diagnosing may have pituitary tumor or radiation.Illness (including, but is not limited to metabolic syndrome, craniocerebral injury, obesity, osteoporosis or dysthymia disorders) may cause the symptom of the similar growth hormone deficiency among the grownup.
Agonist GH (for example, hGH) varient can work to stimulate its immunity system by increasing mammiferous immunologic function, no matter this increase is because antibody is regulated or cell is regulated, and no matter immunity system is for through GH (for example, hGH) host of polypeptide treatment accepts GH (for example, the hGH) host receptor of polypeptide (as in bone marrow transplantation) for endogenic still migrating to from donor." immune disorder " comprise that wherein individual immunity system has and be less than normal immune any illness to antigenic antibody or cell response, includes, but is not limited to those because medicine (for example chemotherapy) treatment and individuality that the spleniculus immunity reduces.The example of suffering from the individuality of immune disorder comprises (for example) gerontal patient, stand chemotherapy or radiocurable individuality, the individuality that grave illness is just healed maybe will be accepted operating individuality, the individuality of suffering from AIDS, suffers from congenital and acquired character B granulocytopenia (such as hypogammag lobulinemia, common variant agammaglobulinaemia and selective immunoglobulin deficiency (for example IgA deficiency disease)) the patient, the patient who infected by virus (its have compared to patient's immune response shorter latent period) such as rabies virus; With the patient who suffers from genetic disorder disease (such as the diGeorge syndromes).
(for example, hGH) the antagonist polypeptide can be used for treating gigantosoma and the reactive malignant tumour of acromegaly, diabetes and the complication (diabetic retinopathy, diabetic neuropathy) that is produced by diabetes, vascular eye (for example comprising the hyperplasia revascularization), ephrosis and GH to GH of the present invention.
Vascular eye comprises (for example) retinopathy (being caused by (for example) anemia of prematurity disease or herrik syndrome) and macular degeneration.
The reactive malignant tumour of GH (for example comprises (for example) Weir Mu Shi tumour (Wilm ' s tumor), sarcoma, osteogenic sarcoma), mammary cancer, colorectal carcinoma, prostate cancer and thyroid carcinoma, and the cancer (that is, the cancer of placenta, thymus gland, brain, sialisterium, prostate gland, marrow, skeletal muscle, tracheae, spinal cord, retina, lymphoglandula and from burkitt's lymphoma (Burkitt ' s lymphoma), colorectal cancer, lung cancer, lymphoid leukemia and melanomatous cancer) of expressing the tissue of GH receptor mrna.
For example, GH of the present invention (for example, hGH) the agonist polypeptide can be used for treating chronic renal failure, the retardation of growth relevant, relevant with Turner's synodrome (Turner ' s Syndrome) of short and small stature, paediatrics Pu Laide-Willie syndromes (Prader-Willi Syndrome) with chronic renal insufficiency (CRI) (PWS), suffer from become thin or cachectic HIV patient, less than children, obesity and the osteoporosis of birth in pregnant age (SGA).
(for example, mean vol hGH) can change GH, and especially should be based on the recommendation and the prescription of qualified physicians.GH (for example, accurate amount hGH) be one about preferred problem, it is arranged by following factor: sanatory accurate type, treatment patient's illness and other composition in the composition.The present invention also provides the dispensing of the another kind of promoting agent of treatment significant quantity.Can easily determine to wait to throw the amount of giving based on the treatment of using hGH by the those skilled in the art.
Medical composition of the present invention can be made with usual manner.
In certain embodiments, the invention provides a kind of methods of treatment, it comprises throws the individuality that gives the needs treatment with the hormonal composition of significant quantity, and described hormonal composition contains the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.In certain embodiments, described method comprises that (for example, hGH) individuality (for example human) is given in throwing with GH.In certain embodiments, GH (for example, hGH) comprises non-naturally encoded amino acid, such as the non-naturally encoded amino acid that contains carbonyl.In certain embodiments, non-naturally encoded amino acid is the amino acid of ketone group containing, for example to acetylphenylalanine.In certain embodiments, GH (for example hGH) contains non-naturally encoded amino acid, for example in GH (for example hGH), replace on the position corresponding to the amino acid 35 among the SEQ ID NO:2 to acetylphenylalanine.Water-soluble polymers can be PEG.Suitable PEG comprises linear PEG and branch PEG; Can use described any PEG herein.In a particular embodiment, PEG is about 0.1 to 100kDa or about 1 to 100kDa or about 10 to 50kDa or about 20 to 40kDa or the linear PEG of about 30kDa.In certain embodiments, medical composition contains the GH (for example hGH) that is connected with the PEG of 30kDa by the oxime key, wherein said oxime key corresponding to the amino acid 35 among the SEQ ID NO:2 locational to acetylphenylalanine and PEG between.In certain embodiments, subject individuality suffer from the paediatrics growth hormone deficiency, special send out property of short and small stature, the Childhood outbreak grownup's growth hormone deficiency, the grownup's growth hormone deficiency or the Secondary cases growth hormone deficiency of Adulthood outbreak.
As described herein and as affiliated field in known, can any suitable form, approach, dosage, frequency and time length (for example, hGH) throw GH to give individuality.In certain embodiments, the invention provides a kind of methods of treatment, it comprises throws the individuality that gives the needs treatment with the hormonal composition of significant quantity, described hormonal composition contains the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, wherein said water-soluble polymers is a linear polymer, and wherein said hormonal composition is every other day only to make an appointment with once, or per 3 days, 4 days, 5 days or 6 days are once, once in a week, per 8 days, 9 days, 10 days, 11 days, 12 days or 13 days are once, whenever biweekly, per 15 days, 16 days, 17 days, 18 days, 19 days or 20 days are once, per three weeks once, per 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days once, every month once or less than mensal approximately frequency administration.Should be appreciated that the dispensing frequency can change according to judgement individual or (more generally) treatment expert, and can use any combination of frequency.In certain embodiments, the GH composition be with weekly only about once, whenever biweekly, per three weeks once or mensal frequency throw and give.In certain embodiments, the GH composition be with weekly only about once, whenever biweekly or mensal frequency throw and give.In certain embodiments, the GH composition is only to make an appointment with frequency throwing once to give weekly.In certain embodiments, the GH composition is to give with the only about frequency throwing once of per two weeks.In certain embodiments, the GH composition is to throw with every month only about frequency once to give.
The present invention also provides the dispensing of the another kind of promoting agent of treatment significant quantity together with hGH of the present invention.Can easily determine to wait to throw the amount of giving based on the treatment of using hGH by the those skilled in the art.
Medical composition of the present invention can be made with usual manner.
Example
Provide following example that the invention that (but not limiting) advocated is described.
Example 1
What this case description was multiple in being used for selecting the potential standard that non-naturally encoded amino acid is incorporated the preferred site of hGH into is provided with is a kind of.
How this examples show selects the preferred site in the hGH polypeptide, to be used to introduce non-naturally encoded amino acid.The crystalline structure 3HHR that will be made up of two molecule compound hGH with acceptor (hGHbp) cell foreign lands is used for determining to introduce one or more non-naturally encoded amino acid whose optimum positions.Utilize (for example, 1AXI) the potential change of the firsts and seconds structural element between the check crystalline structure data set of other hGH structure.The coordinate of these structures can obtain from Protein Data Bank (PDB) people J.Mol.Biol.1997 such as (, 112, the 535 pages) Bernstein, or via www. Rcsb.orgOn The Research Collaboratory for Structural Bioinformatics PDB obtain.Structural model 3HHR contains complete sophisticated 22kDa hGH sequence, but except residue 148 to 153 and the C end F191 residue (because irregular and it is ignored at crystalline structure).There are two disulfide bridge bonds that form by C53 and C165 and C182 and C185.Used in this example sequence numbering is corresponding with the aminoacid sequence of the ripe hGH (22kDa varient) shown in the SEQ ID NO:2.
Use following standard estimation to be used to introduce non-naturally encoded amino acid whose each hGH position: residue (a) should not disturb based on 3HHR, the hGHbp of the structural analysis of 1AXI and 1HWG (with the crystallographic structure of hGHbp monomer or dimer bonded hGH) combines, (b) should not be subjected to the influence (people Science (1989) 243:1330-1336 such as people Science (1989) 244:1081-1085 such as Cunningham and Cunningham) of L-Ala or homologue scanning sudden change, (c) should be that the surface exposes and represent to interact and be minimum with Van der Waals force of residue (van der Waals) or hydrogen bonding on every side, (d) in the hGH varient, should lack or should be variable (Tyr35 for example, Lys38, Phe92, Lys140), (e) behind non-naturally encoded aminoacid replacement, will cause conservative property to change, (f) be found in the highly flexible zone (including, but is not limited to the CD ring) or on the structure in the inflexible zone (including, but is not limited to spiral B).In addition, utilize Cx program people (2002) Bioinformatics such as (, 18, the 980 pages) Pintar to carry out other and calculate, to estimate the protrusion degree of each protein atom at the hGH molecule.Therefore, in certain embodiments, one or more positions in the following column position of (but being not limited to) hGH amino acid that one or more are non-naturally encoded is incorporated into: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191 and 192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid among SEQ ID NO:1 or the SEQ ID NO:3).
In certain embodiments, in one or more following positions: position 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, in one or more following positions: position 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, in one or more following positions: position 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155 (SEQ IDNO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, in one or more following positions: position 30,74,103 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3), with one or more non-naturally encoded aminoacid replacement.In certain embodiments, in one or more following positions: position 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3), with one or more non-naturally encoded aminoacid replacement.
In certain embodiments, the amino acid that the non-natural of one or more positions in including, but is not limited to these positions of following column position exists is connected with water-soluble polymers: position 1 is preceding (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (promptly, at proteinic carboxyl terminal) (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or 3).In certain embodiments, the amino acid that the non-natural of one or more positions under including, but is not limited in these positions of column position exists is connected with water-soluble polymers: position 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3).
In certain embodiments, the amino acid that the non-natural of one or more positions under including, but is not limited in these positions of column position exists is connected with water-soluble polymers: position 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3).
In certain embodiments, the amino acid that the non-natural of one or more positions under including, but is not limited in these positions of column position exists is connected with water-soluble polymers: position 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3).
In certain embodiments, the amino acid that the non-natural of one or more positions under including, but is not limited in these positions of column position exists is connected with water-soluble polymers: position 30,74,103 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3).In certain embodiments, the amino acid that the non-natural of one or more positions in described position exists is connected with water-soluble polymers: position 30,35,74,92,103,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ ID NO:3).In certain embodiments, the amino acid that the non-natural of one or more positions in described position exists is connected with water-soluble polymers: position 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid of SEQ ID NO:1 or SEQ IDNO:3).
Some positions that are used to produce the hGH antagonist comprise: position 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123,127 or the interpolation before position 1, or its any combination (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1, SEQ ID NO:3 or any other GH sequence).Utilize the standard (c)-(e) of agonist design to select these positions.The antagonist design also can comprise the pointed decoration of position I residue, to increase the binding affinity to hGHbp.
Example 2
This example is described the clone and the expression of hGH polypeptide (comprising the non-naturally encoded amino acid in the intestinal bacteria (E.coli)) in detail.This example is also described a kind of bioactive method that is used to assess modified hGH polypeptide.
At United States Patent (USP) the 4th, 601, No. 980; The 4th, 604, No. 359; The 4th, 634, No. 677; The 4th, 658, No. 021; The 4th, 898, No. 830; Detailed description is used to clone hGH and its segmental method in the 5th, 424, No. 199 and the 5th, 795, No. 745, and described patent is to be incorporated herein by reference.Code displaying total length hGH or lack the cDNA of the hGH mature form of N end signal sequence respectively in SEQ ID NO:21 and SEQ ID NO:22.
The introducing translation system that use comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS) is expressed and is contained non-naturally encoded amino acid whose hGH.O-RS preferably makes has the amino acidylate of non-naturally encoded amino acid whose O-tRNA.Described translation system is again in response to coded selection codon, in non-naturally encoded aminoacid insertion hGH.
Table 2:O-RS and O-tRNA sequence.
SEQ?ID NO：4 Tyr Methanococcus jannaschii (M.jannaschii) mtRNACUA tRNA
SEQ?ID NO：5 HLAD03; Optimize amber and suppress tRNA tRNA
SEQ?ID NO：6 HL325A; Optimize the AGGA frameshift suppressor tRNA tRNA
SEQ?ID NO：7 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (6) to azido-L-phenylalanine RS
SEQ?ID NO：8 Be used to incorporate into aminoacyl tRNA synthetase p-BpaRS (1) to benzoyl-L-phenylalanine RS
SEQ?ID NO：9 Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
SEQ?ID NO：10 Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
SEQ?ID NO：11 Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
SEQ?ID NO：12 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (1) to azido--phenylalanine RS
SEQ?ID NO：13 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (3) to azido--phenylalanine RS
SEQ?ID NO：14 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (4) to the triazobenzene L-Ala RS
SEQ?ID NO：15 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (2) to azido--phenylalanine RS
SEQ?ID NO：16 Be used to incorporate into aminoacyl tRNA synthetase (LW1) to ethanoyl-phenylalanine RS
SEQ?ID NO：17 Be used to incorporate into aminoacyl tRNA synthetase (LW5) to ethanoyl-phenylalanine RS
SEQ IDNO：18 Be used to incorporate into aminoacyl tRNA synthetase (LW6) to ethanoyl-phenylalanine RS
SEQ?ID NO：19 Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-5) to azido--phenylalanine RS
SEQ?ID NO：20 Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-6) to azido--phenylalanine RS
With containing modified hGH gene and quadrature aminoacyl tRNA synthetase/tRNA the plasmid transformation escherichia coli (E.coli) of (is specific for the non-naturally encoded amino acid of want) is incorporated into non-naturally encoded amino acid site specific ground in the hGH polypeptide with permission.Through transformed into escherichia coli (E.coli) (in containing 0.01 to 100mM specific non-naturally encoded amino acid whose substratum, growing under 37 ℃) with high frequency high fidelity and the modified hGH of high-efficient expression.Containing non-naturally encoded amino acid whose hGH through the His mark is produced by intestinal bacteria (E.coli) host cell with the form of inclusion body or aggregate.In 6M guanidine HCl, under the sex change condition, described aggregate is dissolved and affinity purification.By under 4 ℃ in 50mM TRIS-HCl, pH8.0,40 μ M CuSO 4Carry out refolding with dialyzed overnight in 2% (w/v) the N-sarcosyl (Sarkosyl).Then with described material to 20mM TRIS-HCl, pH8.0,100mM NaCl, 2mM CaCl 2Dialysis removes the His mark then.Referring to people such as Boissel, (1993) J.Bio.Chem.268:15983-93.The method that is used for the hGH purifying for known, and is to wait by SDS-PAGE, west ink dot analysis or electron spray(ES) ion trap mass spectrometry to confirm for the those skilled in the art.
Fig. 6 is the SDS-PAGE of purified hGH polypeptide.(Invitrogen, Carlsbad CA) via the standard His labelled protein purifying procedure that is provided by manufacturers, come purifying through the mutant hGH of His mark albumen by anion-exchange column then, are loaded on the gel then to use ProBond nickel resin.Colour band 1 shows molecular weight marker, and the N-His hGH of alpha-non-natural amino acid is not incorporated in colour band 2 expressions into.Colour band 3 to 10 contains N-His hGH mutant, and it comprises alpha-non-natural amino acid to acetylphenylalanine on each position in position Y35, F92, Y111, G131, R134, K140, Y143 and K145 respectively.
For further assessing the biological activity of modified hGH polypeptide, use the calibrating that the downstream mark of hGH and its acceptor interaction is measured.Tyrosine phosphorylation takes place in signal transducer and activation (STAT5) that the interaction of the acceptor that hGH and its endogenous produce causes transcribing the family member in human IM-9 lymphocyte series.From IM-9 cDNA storehouse, differentiate two kinds of forms of STAT5, STAT5A and STAT5B.Referring to people such as for example Silva, Mol.Endocrinol. (1996) 10 (5): the 508-518 page or leaf.Because rat growth hormone or human prolactin all do not cause detectable STAT5 phosphorylation, so the human growth hormone's acceptor on the IM-9 cell is optionally for the human growth hormone.Importantly, the pSTAT5 phosphorylation that hGH stimulates is competed effectively with the G120R with hGH in rat GHR (L43R) cell foreign lands.
Stimulate the IM-9 cell with hGH polypeptide of the present invention.Human IM-9 lymphocyte is that (Manassas VA), and makes it be supplemented with Sodium.alpha.-ketopropionate, penicillin, Streptomycin sulphate (Invitrogen available from ATCC, Carlsbad, San Diego) and 10% heat inactivation foetal calf serum (Hyclone, Logan, among the RPMI 1640 UT) growth.The IM-9 cell is being examined and determine a hungry night in the substratum (FBS, Sodium.alpha.-ketopropionate, penicillin and the Streptomycin sulphate through the charcoal/dextran processing that do not contain phenol red RPMI, 10mM Hepes, 1% heat inactivation), and the hGH polypeptide with 12 dose point scopes stimulated 10 minutes under 37 ℃ subsequently.Through irritation cell, change 1 hour thoroughly on ice with 90% ice cold methanol with 1% formaldehyde fixed afterwards.(Cell Signaling Technology, Beverly MA) carried out cell inner dyeing 30 minutes, then dyeed in conjunction with secondary antibody with PE, thereby detected the STAT5 phosphorylation level by at room temperature using elementary phosphorylation STAT5 antibody.Utilize FACSArray to carry out sample collecting, (Tree Star Inc., Ashland OR) analyze the data of being gathered with Flowjo software.Obtain the EC50 value according to the dose response curve that utilizes SigmaPlot to be drawn with the relative protein concn of average fluorescent strength (MFI).
Hereinafter table 3 is summarized the IM-9 data about mutant hGH polypeptide that produced.Use described human IM-9 cell to test the various hGH polypeptide that have the alpha-non-natural amino acid replacement at the different positions place.Specific, Fig. 7, A figure shows the IM-9 data through the hGH of His mark polypeptide, and Fig. 7, B figure shows the IM-9 data of hGH through the His mark (comprise replace Y143 alpha-non-natural amino acid to acetylphenylalanine).The biological activity of the PEGization hGH polypeptide that uses same calibrating to assess to comprise alpha-non-natural amino acid.
Table 3 LE3
GH EC 50(nM) GH EC 50(nM)
WHO?WT 0.4±0.1(n＝8) G120R ＞200,000
N-6His?WT 0.6±0.3(n＝3) G120pAF ＞200,000
Rat GH WT ＞200,000 G131pAF 0.8±0.5(n＝3)
Y35pAF 0.7±0.2(n＝4) P133pAF 1.0
E88pAF 0.9 R134pAF 0.9±0.3(n＝4)
Q91pAF 2.0±0.6(n＝2) T135pAF 0.9
F92pAF 0.8±0.4(n＝9) G136pAF 1.4
R94pAF 0.7 F139pAF 3.3
S95pAF 16.7±1.0(n＝2) K140pAF 2.7±0.9(n＝2)
N99pAF 8.5 Y143pAF 0.8±0.3(n＝3)
Y103pAF 130,000 K145pAF 0.6±0.2(n＝3)
Y111pAF 1.0 ?A155pAF ?1.3
Example 3
This example describe in detail contain the amino acid whose introducing of carbonyl and subsequently with the reaction of the PEG that contains the amino oxygen base.
A kind of method that is used to produce the hGH polypeptide of this examples show is incorporated the non-naturally encoded amino acid of ketone group containing in the described hGH polypeptide, it is approximately 5,000 the PEG that contains the amino oxygen base with molecular weight subsequently and reacts.According to each person in the determined residue 35,88,91,92,94,95,99,101,103,111,120,131,133,134,135,136,139,140,143,145 of the standard of example 1 (hGH) and 155 independently through having the non-naturally encoded aminoacid replacement of following array structure:
Figure A20058004446401931
The sequence that is used for incorporating hGH into to acetylphenylalanine site specific ground is SEQ ID NO:2 (hGH)
Tyr and SEQ ID NO:4 (muttRNA, Methanococcus jannaschii mtRNACUA) and 16,17 or 18 (TyrRS LW1,5 or 6) (above described in the example 2).
In case after modified, comprise contain carbonylamino acid the hGH polypeptide variants promptly with the PEG derivatives reaction that contains the amino oxygen base of following form:
R-PEG(N)-O-(CH 2) n-O-NH 2
Wherein R is a methyl, n be 3 and N be about 5,000 molecular weight.Make with 10mg/mL and be dissolved in 25mM MES (Sigma Chemical, St.Louis, MO) pH6.0,25mM Hepes (Sigma Chemical, St.Louis, MO) pH7.0 or 10mM sodium acetate (Sigma Chemical, St.Louis, MO) the purified hGH and 10 to the 100 times of excessive PEG that contain the amino oxygen base that contain acetylphenylalanine among the pH4.5 react, and then at room temperature stir 10 to 16 hours (Jencks, W.J.Am.Chem.Soc.1959,81, the 475 pages).Then PEG-hGH is diluted in the suitable buffer, for purifying and analysis immediately.
Example 4
Combination with the PEG that comprises the azanol group that is connected with PEG by amido linkage.
Program described in the use-case 3 makes the PEG reagent with following array structure and the non-naturally encoded amino acid coupling of ketone group containing:
R-PEG(N)-O-(CH 2) 2-NH-C(O)(CH 2) n-O-NH 2.
R=methyl wherein, n=4 and N are about 20,000 molecular weight.Described in reaction conditions, purification condition and analysis condition such as the example 3.
Example 5
This example is described in detail two kinds of different non-naturally encoded amino acid is incorporated in the hGH polypeptide.
A kind of method that is used to produce the hGH polypeptide of this example explanation is incorporated in the non-naturally encoded amino acid that two positions in the following residue comprise ketone group functional group: E30, E74, Y103, K38, K41, K140 and K145 in the described hGH polypeptide.Except two different sites places in nucleic acid introduce the selection codon, according to the hGH of preparation described in example 1 and 2 polypeptide.
Example 6
This example is described combining and subsequently in-situ reducing of hGH polypeptide and the PEG that contains hydrazides in detail.
The hGH polypeptide that contains carbonylamino acid is wherein incorporated in preparation into according to the program described in example 2 and 3.In case after modified, the hydrazides PEG that contains with following array structure promptly combines with described hGH polypeptide:
R-PEG(N)-O-(CH 2) 2-NH-C(O)(CH 2) n-X-NH-NH 2，
R=methyl wherein, n=2 and N=10,000 molecular weight, and X is carbonyl (C=O).Make the purified hGH that contains acetylphenylalanine be dissolved in 25mM MES (SigmaChemical with the concentration between the 0.1-10mg/mL, St.Louis, MO) pH6.0,25mM Hepes (Sigma Chemical, St.Louis, MO) pH7.0 or 10mM sodium acetate (Sigma Chemical, St.Louis is MO) among the pH4.5, itself and 1 to 100 times of excessive hydrazides PEG that contains are reacted, and be dissolved in H by interpolation 2Deposit 1M NaCNBH among the O 3(Sigma Chemical, St.Louis MO) make corresponding hydrazone in-situ reducing up to 10 to 50mM ultimate density.In the dark under room temperature, reacted 18 to 24 hours in 4 ℃.(MO) (pH value is about 7.6) comes stopped reaction until the final Tris concentration of 50 mM for Sigma Chemical, St.Louis, or reactant is diluted in the suitable buffer for purifying immediately by adding 1M Tris.
Example 7
This example is described the amino acid that will contain alkynyl in detail and is introduced in the hGH polypeptide and with mPEG-trinitride derivatize.
Following residue: 35,88,91,92,94,95,99,101,131,133,134,135,136,140,143, the 145 and 155 following non-naturally encoded amino acid (hGH that respectively hang oneself; SEQ ID NO:2) replace:
Figure A20058004446401951
The sequence that is used for incorporating hGH into to propargyl tyrosine site specific ground be SEQ ID NO:2 (hGH),
TyrSEQ ID NO:4 (muttRNA, Methanococcus jannaschii mtRNACUA) and 9,10 or 11 (above described in the examples 2).In intestinal bacteria (E.coli), express the hGH polypeptide that contains propargyl tyrosine, and the condition described in the use-case 3 is purified.
Make the purified hGH that contains propargyl tyrosine with 0.1 to 10mg/mL be dissolved in the PB damping fluid (the 100mM sodium phosphate, 0.15M NaCl, pH=8) in, and 10 to 1000 times of excessive PEG that contain azido-are added in the reaction mixture.Follow CuSO with catalytic amount 4Be added in the reaction mixture with the Cu silk.Cultivate described mixture (include, but is not limited under the room temperature or 37 ℃ about 4 hours down, or spend the night under 4 ℃) afterwards, add H 2O and filter described mixture by dialysis membrane.Can be by the addition of this sample of programanalysis described in (including, but is not limited to) example 3.
In this example, PEG will have following array structure:
R-PEG(N)-O-(CH 2) 2-NH-C(O)(CH 2) n-N 3，
Wherein R is a methyl, n be 4 and N be 10,000 molecular weight.
Example 8
This example is described in detail in the medium-and-large-sized hydrophobic amino acid of hGH polypeptide and replaces through propargyl tyrosine.
Be present in Phe, Trp in the zone of one in the column region under the hGH or Tyr residue: 1-5 (N end), 6-33 (spiral A), the 34-74 (zone between spiral A and the spiral B, the A-B ring), 75-96 (spiral B), the 97-105 (zone between spiral B and the spiral C, the B-C ring), 106-129 (spiral C), the 130-153 (zone between spiral C and the spiral D, C-D ring), 154-183 (spiral D), 184-191 (C end) (SEQ ID NO:2), described in example 7 through following non-naturally encoded aminoacid replacement:
Figure A20058004446401961
In case after modified, PEG promptly is connected to and comprises the hGH polypeptide variants that contains alkynyl amino acids.Described PEG will have following array structure:
Me-PEG(N)-O-(CH 2) 2-N 3，
And the coupling program will be followed the program in the example 7.This with generation comprise with naturally occurring large-scale hydrophobic amino acid in a kind of roughly with the non-naturally encoded amino acid whose hGH polypeptide variants of joining (isosteric), and its different sites place in polypeptide is modified through the PEG derivative.
Example 9
This example is specifically described as the generation of hGH homologous peptide dimer, heterodimer, homology polymer or heteropolymer that one or more PEG connexons are separated.
Make the difunctionality PEG derivatives reaction that contains alkynyl hGH polypeptide variants and following form of manufacturing in the example 7:
N 3-(CH 2) n-C(O)-NH-(CH 2) 2-O-PEG(N)-O-(CH 2) 2-NH-C(O)-(CH 2) n-N 3，
Wherein n be 4 and PEG have about 5,000 molecular-weight average, to produce corresponding hGH homologous peptide dimer, wherein separated by PEG on two hGH molecular entities.In a similar fashion, can make hGH polypeptide and one or more other polypeptide coupling, to form heterodimer, homology polymer or heteropolymer.To as example 7 and 3, carry out coupling, purifying and analysis.
Example 10
This example is described the coupling of carbohydrate part and hGH polypeptide in detail.
Residue in the following residue: 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (hGH, SEQ ID NO:2), described in example 3 through following non-naturally encoded aminoacid replacement:
Figure A20058004446401971
In case after modified, comprise the hGH polypeptide variants that contains carbonylamino acid, promptly be connected the reaction of amino oxygen base analogue with the β of N-acetylglucosamine (GlcNAc).HGH polypeptide variants (10mg/mL) and amino oxygen base carbohydrate (21mM) are mixed in water-based 100mM sodium acetate buffer (pH5.5), and cultivated 7 to 26 hours down at 37 ℃.By under envrionment temperature with carbohydrate in conjunction with hGH polypeptide (5mg/mL) and UDP-semi-lactosi (16mM) and β-1,4-galactosyltransferase (galacytosyltransferase) (0.4 unit/mL) in 150mM HEPES damping fluid (pH7.4), cultivated 48 hours together, make second carbohydrate be coupled to first carbohydrate (people J.Biol.Chem.1970 such as Schanbacher in the enzymatic mode, 245,5057-5061).
Example 11
This example is described the generation of PEGization hGH polypeptide antagonist in detail.
Residue in the following residue: 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 or 127 (hGH, SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3), as described in the example 3 through following non-naturally encoded aminoacid replacement.
Figure A20058004446401972
In case after modified, comprise contain carbonylamino acid the hGH polypeptide variants just will with the PEG derivatives reaction that contains the amino oxygen base of following form:
R-PEG(N)-O-(CH 2) n-O-NH 2，
Wherein R is a methyl, n be 4 and N be 20,000 molecular weight, comprise non-naturally encoded amino acid whose hGH polypeptide antagonist with generation, its single position in polypeptide is modified through the PEG derivative.As example 3, carry out coupling, purifying and analysis.
Example 12
Wherein the hGH molecule is the generation of direct-connected hGH homologous peptide dimer, heterodimer, homology polymer or heteropolymer
Comprise the hGH polypeptide variants that contains alkynyl amino acids and can directly be coupled to another and comprise and contain the amino acid whose hGH polypeptide variants of azido-, it is included in the non-naturally encoded aminoacid replacement on the position described in (including, but is not limited to) example 10 separately.This will produce corresponding hGH homologous peptide dimer, and wherein two hGH polypeptide variants connect at II bonding interface place, position entity.In a similar fashion, can make the hGH polypeptide be coupled to one or more other polypeptide, to form heterodimer, homology polymer or heteropolymer.As example 3,6 and 7, carry out coupling, purifying and analysis.
Example 13
PEG-OH+Br-(CH 2) n-C≡CR’→PEG-O-(CH 2) n-C≡CR’
A B
Poly-alkylene glycols (P-OH) and alkylogen (A) are reacted to form ether (B).In described compound, n is that 1 to 9 integer and R ' can be straight or branched, saturated or unsaturated C 1To C 20Alkyl or assorted alkyl.R ' also can be C 3To C 7Saturated or unsaturated cycloalkyl or ring-type mix alkyl, the aryl that is substituted or is unsubstituted or heteroaryl, or the alkaryl that is substituted or is unsubstituted (described alkyl is C 1To C 20Saturated or unsaturated alkyl) or assorted alkaryl.Usually, PEG-OH is for having 800 to 40,000 dalton (Da) molecular weight polyethylene glycol (PEG) or mono methoxy polyethylene glycol (mPEG).
Example 14
mPEG-OH+Br-CH 2-C≡CH→mPEG-O-CH 2-C≡CH
(12mg 0.5mmol) handles and to have 20, the mPEG-OH of 000Da molecular weight (mPEG-OH 20kDa with the NaH among the THF (35mL); 2.0g; 0.1mmol, Sunbio).Then propargyl bromide is dissolved in 80 weight % solution in the dimethylbenzene (0.56mL, 5mmol, 50 equivalents, Aldrich) and the KI of catalytic amount be added in the described solution, and the gained mixture heating up is lasted 2 hours to refluxing.Then add water (1mL) and move down and desolventize in vacuum.In resistates, add CH 2Cl 2(25mL) and with organic layer separate, through anhydrous Na 2SO 4Drying, and volume is reduced to about 2mL.With this CH 2Cl 2Solution dropwise is added in the ether (150mL).Collection gained precipitation is with some parts of cold diethyl ether washings and dry, to obtain propargyl-O-PEG.
Example 15
mPEG-OH+Br-(CH 2) 3-C≡CH→mPEG-O-(CH 2) 3-C≡CH
(12mg 0.5mmol) handles and to have 20, the mPEG-OH of 000Da molecular weight (mPEG-OH 20kDa with the NaH among the THF (35mL); 2.0g; 0.1mmol, Sunbio).Then (Aldrich) KI with catalytic amount is added in the described mixture for 0.53mL, 5mmol with 50 normal 5-bromo-1-pentynes.The gained mixture heating up is lasted 16 hours to refluxing.Then add water (1mL) and move down and desolventize in vacuum.In resistates, add CH 2Cl 2(25mL) and with organic layer separate, through anhydrous Na 2SO 4Drying, and volume is reduced to about 2mL.With this CH 2Cl 2Solution dropwise is added in the ether (150mL).Collection gained precipitation is with some parts of cold diethyl ether washings and dry, to obtain corresponding alkynes.In similar reaction, can use 5-chloro-1-pentyne.
Example 16
(1)m-HOCH 2C 6H 4OH+NaOH+Br-CH 2-C≡CH→m-HOCH 2C 6H 4O-CH 2-C≡CH
(2)m-HOCH 2C 6H 4O-CH 2-C≡CH+MsCl+N(Et) 3→m-MsOCH 2C 6H 4O-CH 2-C≡CH
(3)m-MsOCH 2C 6H 4O-CH 2-C≡CH+LiBr→m-Br-CH 2C 6H 4O-CH 2-C≡CH
(4)mPEG-OH+m-Br-CH 2C 6H 4O-CH 2-C≡CH→mPEG-O-CH 2-C 6H 4O-CH 2-C＝CH
At first to the 3-hydroxy-benzyl alcohol (2.4g, 20mmol) add in the solution in THF (50mL) and water (2.5mL) Powdered sodium hydroxide (1.5g, 37.5mmol), then add propargyl bromide be dissolved in 80 weight % solution in the dimethylbenzene (3.36mL, 30mmol).The reaction mixture refluxed heating is lasted 6 hours.In described mixture, add 10% citric acid (2.5mL) and move down and desolventize in vacuum.With ethyl acetate (3 * 15mL) extracted residues, and with saturated NaCl solution (10mL) washing combination organic layer, through MgSO 4Drying, and concentrate, to obtain 3-propargyloxy benzylalcohol.
Under 0 ℃ with methane sulfonyl chloride (2.5g, 15.7mmol) and triethylamine (2.8mL, (2.0g is 11.0mmol) in CH 20mmol) to be added into compound 3 2Cl 2In solution in, and place refrigerator to last 16 hours the reactant.Common processing (work-up) obtains being the methanesulfonates of light yellow oily.Make described oil (2.4g 9.2mmol) is dissolved among the THF (20mL), and add LiBr (2.0g, 23.0mmol).Reaction mixture is heated to backflow lasts 1 hour, then be cooled to room temperature.In mixture, add water (2.5mL) and move down and desolventize in vacuum.With ethyl acetate (3 * 15mL) extracted residues, and with saturated NaCl solution (10mL) washing combination organic layer, through anhydrous Na 2SO 4Dry and concentrated, wanted bromide to obtain.
With mPEG-OH 20kDa (1.0g, 0.05mmol Sunbio) are dissolved among the THF (20mL), and in ice bath cooling solution.Under the vigorous stirring situation through period of some minutes add NaH (6mg, 0.25mmol), add then the bromide that above obtained (2.55g, 11.4mmol) and the KI of catalytic amount.Remove cooling bath, and the gained mixture heating up is lasted 12 hours to refluxing.In mixture, add water (1.0mL) and move down and desolventize in vacuum.In resistates, add CH 2Cl 2(25mL) and with organic layer separate, through anhydrous Na 2SO 4Drying, and volume is reduced to about 2mL.Dropwise be added into ethereal solution (150mL) and obtain white precipitate, it is collected to obtain the PEG derivative.
Example 17
mPEG-NH 2+X-C(O)-(CH 2) n-C≡CR’→mPEG-NH-C(O)-(CH 2) n-C≡CR’
Also can be coupled to the reactive molecule of the alkynyl functional group shown in containing as mentioned, thereby obtain to contain poly-(ethylene glycol) polymkeric substance of terminal alkynyl by poly-(ethylene glycol) polymkeric substance that will contain functional end-group.N is between 1 and 10.R ' can be H or C 1To C 4Little alkyl.
Example 18
(1)HO 2C-(CH 2) 2-C≡CH+NHS+DCC→NHSO-C(O)-(CH 2) 2-C≡CH
(2)mPEG-NH 2+NHSO-C(O)-(CH 2) 2-C≡CH→mPEG-NH-C(O)-(CH 2) 2-C＝CH
(2.943g 3.0mmol) is dissolved in CH with the 4-pentynoic acid 2Cl 2(25mL).Add N-maloyl imines (3.80g, 3.3mmol) and DCC (4.66g, 3.0mmol), and with solution stirred overnight at room temperature.The thick NHS ester 7 of gained promptly is used for the next step without being further purified.
To have 5, the mPEG-NH of 000Da molecular weight 2(mPEG-NH2,1g Sunbio) are dissolved among the THF (50mL), and mixture is cooled to 4 ℃.Under the vigorous stirring situation by part add a NHS ester 7 (400mg, 0.4mmol).In room temperature, allow mixture was stirred 3 hours in temperature.Then add water (2mL) and move down and desolventize in vacuum.In resistates, add CH 2Cl 2(50mL) and with organic layer separate, through anhydrous Na 2SO 4Drying, and volume is reduced to about 2mL.With this CH 2Cl 2Solution dropwise is added into ether (150mL).Collect gained precipitation and dry under vacuum.
Example 19
This example presents the preparation of the methylsulfonyl ester of poly-(ethylene glycol), and the methylsulfonyl ester of described poly-(ethylene glycol) also can be called as the methane sulfonate or the methanesulfonates of poly-(ethylene glycol).Corresponding tosylate and halogenide can be by similar program preparations.
mPEG-OH+CH 3SO 2Cl+N(Et) 3→mPEG-O-SO 2CH 3→mPEG-N 3
With the mPEG-OH in the 150mL toluene (molecular weight=3,400,25g, 10mmol) component distillation 2 hours under nitrogen, and solution is cooled to room temperature.With the anhydrous CH of 40mL 2Cl 2(15mmol) is added in the described solution with the 2.1mL anhydrous triethylamine.In ice bath the cooling described solution, and dropwise add 1.2mL through distillatory methane sulfonyl chloride (15mmol).With solution stirred overnight in nitrogen atmosphere at room temperature, and come stopped reaction by adding the 2mL dehydrated alcohol.Under vacuum, mixture is evaporated to remove solvent (being mainly toluene solvent in addition), filter, under vacuum, concentrate once more, then be precipitated in the 100 mL ether.With some parts of cold diethyl ether wash filtrates and dry in a vacuum, to obtain methanesulfonates.
(20g 8mmol) is dissolved among the 75ml THF, and solution is cooled to 4 ℃ with methanesulfonates.To in cooling solution, add sodiumazide (1.56g, 24mmol).Under nitrogen, reactant is heated to backflow and lasts 2 hours.Follow evaporating solvent and use CH 2Cl 2(50mL) dilution resistates.With NaCl solution washing organic fraction, and through anhydrous MgSO 4Dry.Volume is reduced to 20mL, and makes the product precipitation in the cold anhydrous ether of 150mL by adding to.
Example 20
(1)N 3-C 6H 4-CO 2H→N 3-C 6H 4CH 2OH
(2)N 3-C 6H 4CH 2OH→Br-CH 2-C 6H 4-N 3
(3)mPEG-OH+Br-CH 2-C 6H 4-N 3→mPEG-O-CH 2-C 6H 4-N 3
Can use United States Patent (USP) the 5th, 998, the method described in No. 595 is made 4-azido-benzylalcohol, and described patent is to be incorporated herein by reference.Under 0 ℃ with methane sulfonyl chloride (2.5g, 15.7mmol) and triethylamine (2.8mL, (1.75g is 11.0mmol) in CH 20mmol) to be added into 4-azido-benzylalcohol 2Cl 2In solution in, and place refrigerator to last 16 hours the reactant.Common processing obtains being the methanesulfonates of light yellow oily.Described oil (9.2mmol) is dissolved among the THF (20mL), and interpolation LiBr (2.0g, 23.Ommol).Reaction mixture is heated to backflow lasts 1 hour, then be cooled to room temperature.In mixture, add water (2.5mL) and move down and desolventize in vacuum.With ethyl acetate (3 * 15mL) extracted residues, and with saturated NaCl solution (10mL) washing combination organic layer, through anhydrous Na 2SO 4Dry and concentrated, wanted bromide to obtain.
With the NaH among the THF (35mL) (12mg, 0.5mmol) handle mPEG-OH 20kDa (2.0g, 0.1mmol, Sunbio), and with bromide (3.32g, 15mmol) KI together with catalytic amount is added in the mixture.The gained mixture heating up is lasted 12 hours to refluxing.In mixture, add water (1.0mL) and move down and desolventize in vacuum.In resistates, add CH 2Cl 2(50mL) and with organic layer separate, through anhydrous Na 2SO 4Drying, and volume is reduced to about 2mL.Dropwise be added into and obtain precipitation in the ethereal solution (150mL), it is collected to obtain mPEG-O-CH 2-C 6H 4-N 3
Example 21
NH 2-PEG-O-CH 2CH 2CO 2H+N 3-CH 2CH 2CO 2-NHS→N 3-CH 2CH 2-C(O)NH-PEG-O-CH 2CH 2CO 2H
With NH 2-PEG-O-CH 2CH 2CO 2(molecular weight 3,400Da 2.0g) are dissolved in saturated NaHCO to H 3In the aqueous solution (10mL), and solution is cooled to 0 ℃.Under the vigorous stirring situation, add 3-azido--1-N-maloyl imido grpup propionic ester (5 equivalent).After 3 hours, add 20ml H 2O and at room temperature with mixture restir 45 minutes.Use 0.5N H 2SO 4The pH value is adjusted to 3, and adds the concentration of NaCl until about 15 weight %.Use CH 2Cl 2(100mL * 3) extractive reaction mixture is through Na 2SO 4Dry and concentrated.Using the cold diethyl ether post precipitation, by filtration collection product and dry under vacuum, to obtain ω-carboxyl-azide PEG derivative.
Example 22
mPEG-OMs+HC≡CLi→mPEG-O-CH 2-CH 2-C≡C-H
Under the vigorous stirring situation, in the solution (4 equivalent) of acetylene lithium in THF (known in as affiliated field and prepare, and through being cooled to-78 ℃), dropwise add the mPEG-OM solution that is dissolved among the THF.After 3 hours, allow with the reactant temperature to room temperature and by adding the 1mL butanols stopped reaction.Then add 20mL H 2O, and at room temperature with mixture restir 45 minutes.Use 0.5N H 2SO 4The pH value is adjusted to 3, and adds the concentration of NaCl until about 15 weight %.Use CH 2Cl 2(100mL * 3) extractive reaction mixture is through Na 2SO 4Dry and concentrated.Using the cold diethyl ether post precipitation, by filtration collection product and dry under vacuum, to obtain 1-(fourth-3-alkynyloxy base)-methoxy poly (ethylene glycol) (mPEG).
Example 23
The amino acid that uses the method described in the following document will contain azido-is optionally incorporated in the protein with the amino acid position that contains ethynyl: people such as L. Wang, (2001), Science292:498-500; People such as J.W.Chin, Science301:964-7 (2003)); People such as J.W.Chin, (2002), Journal of the American Chemical Society124:9026-9027; J.W.Chin ， ﹠amp; P. G. Schultz, (2002), Chem Bio Chem3 (11): 1135-1137; People such as J.W.Chin, (2002), PNAS United States of America99:11020-11024; With L. Wang ， ﹠amp; P.G.Schultz, (2002), Chem.Comm.,1:1-11.In case after incorporating amino acid into, promptly under 37 ℃, in the pH value is 8 phosphate buffered saline buffer (PB), have 2mM PEG derivative, 1mM CuSO 4With carry out proteinic cycloaddition reaction under the situation of about 1mg Cu silk with 0.01mM, last 4 hours.
Example 24
This case description is to ethanoyl-D, L-phenylalanine (pAF) and m-PEG-hydroxylamine derivative synthetic.
Use before at Zhang Z., Smith, B.A.C, Wang, L., Brock, A., Cho, C.﹠amp; Schultz, P.G., Biochemistry, the program described in (2003) 42, the 6735-6746 is synthesized racemize pAF.
Finish follow procedure with synthetic m-PEG-hydroxylamine derivative.To (N-tertbutyloxycarbonyl-amino oxygen base) acetate (0.382g, 2.0mmol) and 1, (0.16mL, 1.0mmol) (DCM 70mL) adds methoxyl group-polyoxamide (m-PEG-NH to the 3-DIC in the solution in (it at room temperature is stirred 1 hour) in methylene dichloride 2, 7.5g, 0.25mmol, Mt.30 K is available from BioVectra) and diisopropylethylamine (0.1mL, 0.5mmol).Reaction product was at room temperature stirred 48 hours, then be concentrated into about 100mL.Mixture dropwise is added in the cold ether (800mL).Tertbutyloxycarbonyl protection product precipitation is separated out, and collected, with ether (3 * 100mL) washings by filtering.By dissolving and in ether (800mL), precipitate twice it is further purified again in DCM (100mL).Dry in a vacuum, obtain 7.2g (96%) product, this confirms by NMR and Nihydrin test.
The tertiary butyloxycarbonyl radical reaction that goes of the protected product (7.0g) that is obtained above carrying out in 50%TFA/DCM (40mL) under 0 ℃ lasts 1 hour, then carries out under room temperature 1.5 hours.After removing most of TFA in a vacuum, be added into the tfa salt that makes hydroxylamine derivative in the resistates by the 4N HCl in diox (1mL) and be converted into HCl salt.With resolution of precipitate in DCM (50mL), and in ether (800mL) once more the precipitation.Collect final product (6.8g, 97%) by filtering, (3 * 100mL) washings, drying is stored in it under nitrogen in a vacuum with ether.Use same program synthetic other PEG (5K, 20K) hydroxylamine derivative.
Example 25
This case description is used to comprise the hGH polypeptide expression method and the purification process of alpha-non-natural amino acid.Quadrature tRNA, quadrature aminoacyl tRNA synthetase and hGH construction transformed host cell have been used.
At first growth in the 2ml defined medium that has 100 μ g/ml penbritins (ampicillin) under 37 ℃ (being supplemented with the glucosyl group basal culture medium of leucine, Isoleucine, trace-metal and VITAMIN) of a little stab culture (stab) of the freezing glycerine reserve that making hangs oneself transforms DH10B (fis3) cell.Work as OD 600Reach at 2 to 5 o'clock, 60ul is transferred to 60ml has in the fresh defined medium of 100 μ g/ml penbritins, and under 37C, grow to 2 to 5 OD once more 600The 50ml culture is transferred in the defined medium that in 5 liters of fermentor tanks (Sartorius BBI) 2 liters have 100 μ g/ml penbritins.With salt of wormwood fermentor tank pH value is controlled at pH6.9, temperature is controlled at 37 ℃, and air flow rate is 51pm, and with polyalkylene defoamer KFO F1 19 (Lubrizol) control foam.The speed of automatically regulating agitator to be keeping dissolved oxygen levels greater than 30%, and if the speed of agitator reach its maximum value, use purity oxygen to replenish air spray so.At 37 ℃ after following 8 hours, with the culture of exponential 50 times of concentration of increment rate feed-in defined medium, to keep 0.15 hour -1Specific growth rate.Work as OD 600Reach at about 100 o'clock, add the racemic mixture of acetylphenylalanine ultimate density, and cool the temperature to 28 ℃ to 3.3mM.After 0.75 hour, add the ultimate density of sec.-propyl-b-D-sulfo-galactopyranoside to 0.25mM.Make cell 28 ℃ of following regrowths 8 hours, granulation, and freezing until further processing down at-80 ℃.
(Invitrogen, Carlsbad CA), via the standard His labelled protein purifying procedure that specification sheets provided by Invitrogen, by anion-exchange column, come purifying through the mutant hGH of His mark albumen then to use ProBond nickel resin.
Purified hGH is concentrated into 8mg/ml, and with damping fluid change into reaction buffer (the 20mM sodium acetate, 150mM NaCl, 1mM EDTA, pH4.0).PEG with 20: 1: the hGH mol ratio is added into MPEG-oxyamine powder in the hGH solution.Softly react under the situation of vibration at 28 ℃ in addition and to last 2 days.Via anion-exchange column from unreacted PEG and hGH purifying PEG-hGH.
Assess the quality of each PEGization mutant hGH by three kinds of calibratings, enter experimentation on animals then.By carrying out the purity that PEG-hGH is checked in 4% to 12% acrylamide NuPAGEBis-Tris gel electrophoresis with MES SDS electrophoretic buffer down in irreducibility condition (Invitrogen).With Coomassie blue (Coomassie blue) with gel-colored.According to photodensitometry scanning, the purity of PEG-hGH band is greater than 95%.Use available from Charles River Laboratories (Wilmington, KTA MA) by kinetics LAL calibrating 2Cover is organized the level of endotoxin of testing each PEG-hGH, and it is less than 5 EU/ dosage.Use the biological activity of IM-9 pSTAT5 bioassay (in example 2, mentioning) assessment PEG-hGH, and EC 50Value is less than 15nM.
Example 26
This case description is used to assess the purifying and the homogeneous method of the hGH polypeptide that comprises alpha-non-natural amino acid.
Fig. 8 is the SDS-PAGE that comprises the hGH polypeptide of alpha-non-natural amino acid on position 92.The colour band 3,4 and 5 of gel is presented at and comprises the covalently bound hGH to acetylphenylalanine to 5kDa, 20kDa or 30kDa PEG molecule on the position 92.Show that in Figure 11 other comprises the PEGization hGH polypeptide of alpha-non-natural amino acid.Each PEG-hGH protein of 5 μ g is loaded on each SDS-PAGE.Figure 11, A figure: colour band 1, molecular weight marker; Colour band 2, WHO rhGH reference standard (2 μ g); Colour band 3 and 7,30KPEG-F92pAF; Colour band 4,30KPEG-Y35pAF; Colour band 5,30KPEG-R134pAF; Colour band 6,20KPEG-R134pAF; Colour band 8, WHO rhGH reference standard (20 μ g).Figure 11, B figure: colour band 9, molecular weight marker; Colour band 10, WHO rhGH reference standard (2 μ g); Colour band 11,30KPEG-F92pAF; Colour band 12,30KPEG-K145pAF; Colour band 13,30KPEG-Y143pAF; Colour band 14,30KPEG-G131pAF; Colour band 15,30KPEG-F92pAF/G120R; Colour band 16, WHO rhGH reference standard (20 μ g).Fig. 9 shows the biological activity of PEGization hGH polypeptide (5kDa, 20kDa or 30kDa) in the IM-9 cell; Method is to carry out described in example 2.
The purity of hGH-PEG binding substances can be carried out mass spectroscopy then by proteolytic degradation (including, but is not limited to the trypsinase cracking) and be assessed that (Pepinsky RB. waits the people, J.Pharmcol.﹠amp; Exp.Ther.297 (3): 1059-66 (2001)).The method that is used to carry out tryptic digestion is also European Pharmacopoeia (2002) the 4th edition, description arranged in the 1938th page.Described method is made amendment.With sample dialyzed overnight in 50mM TRIS-HCl (pH7.5).With 66: 1 mass ratioes rhGH polypeptide and Regular Insulin (through the trypsinase that TPCK handles, Worthington) were cultivated 4 hours in 37 ℃ of water-baths together.Sample is cultivated some minutes on ice, to end digestion reaction and during HPLC analyzes, to maintain 6 ℃ subsequently.To be loaded on 25 * 0.46cm Vydac C-8 post (5 μ m particle diameters, 100  apertures) in 0.1% trifluoroacetic acid through the sample (about 200 μ g) of digestion, and 30 ℃ down with the acetonitrile of 0 to 80% gradient through 70 minutes flow rate wash-outs with 1ml/ minute.Wash-out by the monitoring of the light absorption ratio under 214nm tryptic peptide.
Figure 10, the primary structure of A figure diagram hGH is wherein indicated trypsinase cracking position and with arrow non-natural aminoacid replacement F92pAF (revising the figure of gained according to people Biotechnol Appl Biochem. (1988) 10 (4): 326-337 such as Becker) is described.B figure shows by comprising peptide (the 30K PEG His that non-naturally encoded amino acid whose PEGization hGH polypeptide produces 6-F92pAF rhGH is labeled as A), by comprising the peptide (His that non-naturally encoded amino acid whose hGH polypeptide produces 6-F92pAF rhGH is labeled as B) and the synergetic trypsinase figure of the peptide (WHO rhGH is labeled as C) that produces by wild-type hGH.WHO rhGH and His 6The trypsinase figure of-F92pAF rhGH more only shows two peak shifts (peptide peak 1 and peptide peak 9), and remaining peak is identical.These differences are by through expressing His 6His on the N end of-F92pAF rhGH 6Addition caused, it causes peak 1 displacement; And the displacement at peak 9 is replaced acetylphenylalanine by warp on the residue 92 and causes.C figure shows the amplification from the peak 9 of B figure.His 6-F92pAF and 30K PEG His 6-F92pAF rhGH trypsinase figure relatively is presented at His 6The PEGization postpeak 9 of-F92pAF rhGH disappears, and confirms that thus described modification is specific for peptide 9.
Example 27
This case description is by two formed homodimers of hGH polypeptide of each self-contained alpha-non-natural amino acid.
Figure 12 will comprise the homodimer of this modified polypeptide that links together through the hGH of His mark polypeptide with the functional group with the PEGization that is used for hGH described in the example 25 and reactive difunctionality connexon that acetylphenylalanine is replaced on position 92 IM-9 verification result compares.
Example 28
This case description serves as the monomer and the dimer hGH polypeptide of hGH antagonist.
Wherein G120R replace is introduced among the II of position the hGH mutein can with single hGH receptors bind, but can not make two receptor dimerizations.Described mutein may occupy the receptor site under the situation by the path of posting a letter in active cells not, thereby serves as sv hGH antagonist (Fuh, people Science 256:1677-1680 (1992) such as G.).Figure 13, A figure show to measure by the hGH with G120R replacement makes the IM-9 of pSTAT5 phosphorylation examine and determine data.As Figure 13, shown in the B figure, the hGH polypeptide with alpha-non-natural amino acid that same position (G120) locates to incorporate into causes also can serving as the molecule of hGH antagonist.With Figure 13, the dimer of the hGH antagonist shown in the B figure is configured to functional group and reactive difunctionality connexon with the PEGization that is used for hGH described in the example 25 and links together.Figure 14 shows that this dimer also lacks biological activity in the IM-9 calibrating.
Carry out other calibrating with hGH polypeptide that relatively comprises the G120pAF replacement and the dimer that is connected by the PEG connexon through G120pAF modification hGH polypeptide.Monomer in the dose response scope and the dimer that is connected by pEG connexon competition WHO hGH inductive STAT5 phosphorylation.Also carry out surface receptor competition research, combine with the cell surface receptor of GH competition on IM-9 and rat GHR (L43R)/BAF3 cell with displaying monomer and dimer.Described dimer serves as than the more effective antagonist of monomer.
Table 4 shows the data from described research.
Table 4
Clone IM-9 IM-9 Rat GHR (L43R)/BAF3
Calibrating The inhibition of pSTAT5 The surface receptor competition The surface receptor competition
IC 50(nM) IC 50(nM) IC 50(nM)
The G120pAF monomer 3.3 8.4 3.1
(G120pAF) dimer, the PEG connexon 0.7 2.7 1.4
Example 29
This example detailed description hGH activity and hGH polypeptide are for the measurement of hGH receptor affinity.
The clone and the purifying of rat GH acceptorBetween that the cell foreign lands (GHR ECD, amino acid S29-T238) of rat GH acceptor are cloned into pET20b carrier (Novagen) and the Nde I and Hind III position of C end 6His mark with frame.The sudden change of introducing L43 to R is with further near human GH receptor binding site (people such as Souza, ProcNatl Acad Sci U S A. (1995) 92 (4): 959-63).By under 30 ℃, inducing 4 to 5 hours, thereby in BL21 (DE3) intestinal bacteria (E.coli) cells (Novagen), produce recombinant protein with 0.4mM IPTG.After cytolysis, wash four times by resuspending in Du Ensi tissue homogenizer (dounce) with the 50mM Tris of 30mL (pH 7.6), 100mM NaCl, 1mM EDTA, 1%Triton X-100, and with same buffer (not containing Triton X-100) washed twice.At this, inclusion body is made up of the GHR ECD more than 95%, and it is dissolved in 0.1M Tris (pH11.5), the 2M urea.Pass through S100 (Sigma) gel-filtration column (through 50mM Tris (pH 7.8), 1M L-arginine, 3.7mM cystamine, 6.5mM cysteamine balance) by means of the aliquots containig that makes inclusion body solution, thereby finish refolding.Make the flow point combination that contains soluble protein and to 50mM Tris (pH7.6), 200mM NaCl, the dialysis of 10% glycerine.Tout court with the sample centrifugation removing any precipitation, and cultivate with the aliquots containig of Talon resin (Clontech) according to manufacturer specification.After washing, in dialysis buffer liquid, use 120mM imidazoles elute protein with the 20 volume dialysis buffer liquid that are supplemented with the 5mM imidazoles.Finally, for 50mM Tris (pH7.6), 30mM NaCl, 1mM EDTA, 10% glycerine dialyzed overnight, centrifugation is adjusted to 20% final glycerol concentration to remove any precipitation tout court, in-80 ℃ of following five equilibriums and storage with sample.Use extinction coefficient epsilon=65 as calculated, 700M -1* μ m -1Measure protein concn by OD (280).
The bonded Bio core of GH to GHR TMAnalyze
Use the standard amine coupling program of recommending, the soluble g HR ECD of about 600 to 800 RU is fixed in Biacore as manufacturers TMOn the CM5 chip.Even most of acceptor is by this technology inactivation, finds maximum specificity GH that the immobilization of this degree also is enough to produce about 100 to 150 RU in conjunction with response by experimental technique, and on binding kinetics, do not have noticeable change.Referring to people J Mol Biol. (1993) 234 (3): 554-63 and WellsJA.Proc Natl Acad Sci USA (1996) 93 (1) such as for example Cunningham: 1-6).
With HBS-EP damping fluid (Biacore TM, Pharmacia) in the wild-type mutant GH (0.1 to 300nM) of various concentration last 4 to 5 minutes with 40 μ l/ minutes flow rate and be injected on the GHR surface, and after injection to division monitoring 15 minutes.4.5M MgCl by 15 pulse per second (PPS)s 2Make surface regeneration.After at least 100 reprocessing cycle, only observe minimum binding affinity loss (1% to 5%).The reference cell of sessile receptor is not used to deduct any damping fluid volume effect and non-specific binding.
Use BiaEvaluation 4.1 software (BIACORE TM) handle the kinetics binding data that obtains by the GH titration experiments." divalence analyte " conjunctive model provides gratifying configuration (fit) (chi 2Value is usually less than 3), with continuous 1: 2 (GH: GHR) dimerisation (the Wells JA.Proc Natl Acad Sci U S A (1996) 93 (1): 1-6) that conforms to of suggestion.With balance fission constants (Kd) as indivedual rate constant (k Off/ k On) calculate.
Table 5 demonstration uses the rat GHR ECD (L43R) that is fixed on the CM5 chip from Biacore TMIncorporating parametric.
Table 5
GH k on，×?10 -51/M*s k off，×?10 4，1/s ?K d，nM
WHO?WT 6.4 3.8 ?0.6
N-6His?WT ?9 5.6 ?0.6
Rat GH WT 0.33 83 250
N12pAF 12.5 4.6 0.4
R16pAF 6.8 4.8 0.7
Y35pAF 7.8 5.3 0.7
E88pAF 6.8 5.4 0.8
Q91pAF 6.6 4.9 0.7
F92pAF 8.6 5.0 0.6
R94pAF 5.6 6.0 1.1
S95pAF 0.7 3.1 4.3
N99pAF 2.2 3.8 1.7
Y103pAF About 0.06 About 6 ＞100
Y111pAF 8.4 4.8 0.6
G120R 2.2 22 10
G120pAF 1.1 23 20
G131pAF 6.0 5.3 0.9
P133pAF 6.4 4.9 0.8
R134pAF 8.4 5.8 0.7
T135pAF 7.2 4.5 0.6
G136pAF 6.2 4.3 0.7
F139pAF 6.8 4.4 0.7
K140pAF 7.2 3.7 0.5
Y143pAF 7.8 6.7 0.9
K145pAF 6.4 5.0 0.8
A155pAF 5.8 4.4 0.8
F92pAF-5KD?PEG 6.2 2.3 0.4
F92pAF-20KD?PEG 1.7 1.8 1.1
F92pAF-30KD?PEG 1.3 0.9 0.7
R134pAF-5KD?PEG 6.8 2.7 0.4
R134pAF-30KD?PEG 0.7 1.7 2.4
Y35pAF-30KD?PEG 0.9 0.7 0.7
(G120pAF) dimer 0.4 1.5 3.4
(F92pAF) dimer 3.6 1.8 0.5
The GHR stable cell lines
IL-3 dependency mouse cell lines (BAF3) is passed through in regulating substratum at RPMI 1640, Sodium.alpha.-ketopropionate, penicillin, Streptomycin sulphate, 10% heat inactivation foetal calf serum, 50 μ M 2 mercapto ethanols with as the 10%WEHI-3 clone in IL-3 source in a usual manner.The all cells culture all remains on 37 ℃ of following 5%CO 2Humid atmosphere in.
Use BAF3 clone to set up rat GHR (L43R) stabilized cell clone, 2E2-2B12-F4.In brief, the linearizing pcDNA3.1 plastid that contains total length rat GHR (L43R) cDNA with 15 μ g makes 1 * 10 7The BAF3 cell electropolarization that individual moderate merges.Permission recovered 48 hours through transfectional cell, then cloned by restricted dilution in the substratum that contains 800 μ g/ml G418 and 5nM WHO hGH.GHR expresses transfectant by using anti-human GHR antibody (R ﹠amp; D Systems, Minneapolis, padding MN) is discerned, and (BDBiosciences, San Diego CA) go up analysis at FACS Array.Then screen the transfectant of expressing good GHR grade according to proliferation activity to WHO hGH in the BrdU propagation calibrating (described in hereinafter).Rat GHR (L43R) cell clone of stable transfection build on the transfectant of wanting in the presence of 1.2mg/ml G418 and 5nM hGH, have that surface receptor is expressed and the other two-wheeled of the constant profile of multiplication capacity repeats subclone.The cell clone of Jian Liing (2E2-2B12-F4) is stored in the BAF3 substratum (adding 1.2mg/mlG418 under the situation of no hGH) in a usual manner thus.Propagation by the BrdU mark
Serum starvation rat GHR (L43R) is expressed BAF3 clone (2E2-2B 12-F4) with 5 * 10 4The density of individual cells/well is applied in 96 porose discs.HGH albumen with 12 dose point scopes makes cell activation, and (Sigma, St.Louis MO) carry out mark to use 50 μ MBrdU simultaneously.Cultivate after 48 hours, at room temperature use BD cell fixation/cell permeabilization (cytofix/cytoperm) solution (BD Biosciences) of 100 μ l to make cell fixation/change 30 minutes thoroughly.For exposing the BrdU epitope, the Dnase (Sigma) with 30 μ g/ holes under 37 ℃ will handle 1 hour through the cell of fixing/saturatingization.Use APC to be implemented in sample analysis on the FACS Array in conjunction with the fluorescent immunity inspection dyeing of anti-BrdU antibody (BD Biosciences).
Table 6 shows the biological activity as the PEG hGH mutant of being summarized in pSTAT5 (IM-9) and the calibrating of BrdU propagation.For the comparison between the calibrating, WHO hGH is expressed as consistent.
Table 6
hGH pSTAT5?EC 50(nM) Propagation EC 50(nM)
WHO?WT 1.0 1.0
Y35pAF 1.3 1.6±0.8(n＝3)
Y35pAF-30KPEG 10 5.4±2.8(n＝4)
Y35pAF-40KPEG 53.3 24.0+11.0(n＝3)
F92pAF 2.2±0.4(n＝9) 1.4±0.7(n＝4)
F92pAF-5KPEG 5.1+0.4(n＝3) ND
F92n?AF-20KPEG 10.5+0.8(n＝3) ND
F92pAF-30KPEG 8.8±1.2(n＝8) 4.1±0.9(n＝3)
F92pAF/G120R ＞200,000 ＞200,000
F92pAF/G120R-30K PEG ＞200,000 ＞200,000
G131pAF 2.3±1.8(n＝2) 2.1±1.1(n＝3)
G131pAF-30KPEG 23.8±1.7(n＝2) 4.6±2.4(n＝3)
R134pAF 1.1±0.2(n＝2) 1.7±0.3(n＝3)
R134pAF-20KPEG 5.3 ND
R134pAF-30KPEG 11.3±1.1(n＝2) 2.5±0.7(n＝4)
Y143pAF 1.6±0.1(n＝2) 1.8±0.6(n＝2)
Y143pAF-30KPEG 12.3±0.9(n＝2) 6.6±2.7(n＝3)
K145pAF 2.3±0.5(n＝2) 3.0±1.4(n＝2)
K145pAF-30KPEG 20.6±9.8(n＝2) 5.3±3.5(n＝3)
Example 30
This case description is used to measure the active in vitro and in vivo method of PEGization hGH.
Cell is in conjunction with calibrating
There is not or exists various concentration (volume: 10 μ l) under the situation of unmarked GH, hGH or GM-CSF, and exist 125Under the situation of I-GH (about 100,000cpm or 1ng), under 0 ℃ with two parts of cells (3 * 10 6) 90 minutes (cumulative volumes: 120 μ l) of cultivation in PBS/1%BSA (100 μ l).Cell is suspended and layering again by the ice-cold FCS of 200 μ l in 350 μ l plastics centrifugal separating tubes, and centrifugation (1000g; 1 minute).Collect bead by the end that cuts off pipe, and in gamma counter (Packard) to bead and supernatant liquor separate counts.
Total binding (mean value of replica) under existing according to uncontested person deducts 100 times of excessive unmarked GH and exists combinations (non-specific binding) down (cpm) to measure specificity combination (cpm).Each used cell type is measured non-specific binding.Use identical preparation 125I-GH is not experimentizing on the same day, and should show inherent consistence. 125The I-GH explanation combines with the cell that produces the GH acceptor.Described combination is limited by unmarked natural GH or hGH in the mode of deciding on dosage, but is not subjected to GM-CSF or other passive control restriction.The hGH competition is natural 125I-GH (being similar to natural GH) bonded ability hint, acceptor can fully be discerned two kinds of forms comparably.
The in vivo research of PEGization hGH
Mouse or rat throwing are given PEG-hGH, not modified hGH and buffering solution.The result will show that PEGization hGH of the present invention is compared to superior activity and the persistent transformation period of the not modified hGH that represents by significantly putting on weight.
In conjunction with and non-binding hGH and the measurement of transformation period in vivo of its varient
All experimentation on animalies are all carried out in the equipment of AAALAC approval and under the agreement of the InstitutionalAnimal Care and Use Committee of St.Louis University approval.Rat inhabits individually to have in 12 hours cages in bright/dark circular chamber.For animal provides through the Purina feed 5001 of calibrating and endless water.For the rat that hypophysectomizes, tap water contains 5% glucose extraly.
Pharmacokinetics research
Assess the quality of PEGization mutant hGH by three kinds of calibratings, enter experimentation on animals subsequently.By (Invitrogen, Carlsbad CA) carry out the purity that PEG-hGH is checked in 4% to 12% acrylamide NuPAGE Bis-Tris gel electrophoresis with MES SDS electrophoretic buffer down in the irreducibility condition.With Coomassie blue (Coomassie blue) with gel-colored.According to photodensitometry scanning, the purity of PEG-hGH band is greater than 95%.Use available from Charles River Laboratories (Wilmington, KTA MA) by kinetics LAL calibrating 2Cover is organized the level of endotoxin of testing each PEG-hGH, and it is less than 5 EU/ dosage.Use the biological activity of IM-9 pSTAT5 bioassay (described in example 2) assessment PEG-hGH, and EC 50Value is through confirming less than 15nM.
The pharmacokinetics of the growth hormone compound that will modify through PEG is compared to each other, and compares with the intravital non-PEGization tethelin of male Sprague-Dawley rat (261 to 425g) that obtains from Charles RiverLaboratories.With the surgery modus operandi conduit is installed in carotid artery, is used for blood collecting.After the conduit of success is installed, animal is dispensed to treatment group (3 to 6 every group), administration subsequently.According to 1mg/kg compound (counting 0.41-0.55ml/kg) with dose volume to the animal subcutaneous administration., and gather to the micro-centrifuge tube that applies EDTA via the inlying catheter blood sample collection at a plurality of time points.After centrifugation, gather blood plasma, and store until analysis down at-80 ℃.Use available from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX) antibody sandwich tethelin ELISA cover group is measured compound concentration.Use and administration analogue corresponding standard calculating concentration.Use modeling program WinNonlin (Pharsight, 4.1 editions) estimation pharmacokinetic parameter.Use have linear increases/logarithm minimizing (linear-up/log-down) trapezoidal integration continuously every analysis, and to concentration data weighting equably.
Figure 15 be presented in the rat body behind the single subcutaneous administration average (+/-S.D.) plasma concentration.To rat (every group 3 to 4 of n=) 1mg/kg hGH wild-type protein (WHO hGH) is provided, through His mark hGH polypeptide (his-hGH) or on position 92, comprise the single bolus dosage through His mark hGH polypeptide (30KPEG-pAF92 (his) hGH) of covalently bound alpha-non-natural amino acid to 30kDa PEG to acetylphenylalanine.According to the indicated timed interval and collection plasma sample, and examine and determine described injection compound.30KPEG-pAF92 (his) hGH has compared to the circulation of the remarkable expansion of control hGH.
Figure 16 be presented in the rat body behind the single subcutaneous administration average (+/-S.D.) plasma concentration.(every group 3 to 6 of n=) provides the proteinic single bolus of 1mg/kg dosage to rat.To on each position in 6 different positionss, comprise covalently bound alpha-non-natural amino acid to 30kDa PEG to the hGH polypeptide of acetylphenylalanine and WHO hGH and (his)-hGH compares.According to the indicated timed interval and collection plasma sample, and examine and determine described injection compound.Table 7 shows the values of pharmacokinetic parameters of the dispensing of hGH polypeptide single dose shown in Figure 16.By continuously every the curve of analyzing (Pharsight, 4.1 editions) estimated concentration relative time.Institute's indicating value is mean value (+/-standard deviation).Cmax: peak concentration; Terminal T1/2: the terminal transformation period; AUC 0-〉inf: at the area that is extrapolated under the infinitely-great concentration-time curve; MRT: mean residence time; Cl/f: apparent total plasma clearance; Vz/f: the apparent volume that distributes during the phase not.
Table 7: to the values of pharmacokinetic parameters of common male Sprague-Dawley rat single dose 1mg/kg bolus subcutaneous administration.
Compound (n) Parameter
Cmax (ng/ml) Terminal t 1/2(h) AUC 0->inf(ngXhr/ ml) MRT(h) Cl/f (ml/hr/ kg) Vz/f (ml/kg)
WHO?hGH(3) 529 (±127) 0.53 (±0.07) 759(±178) 1.29 (±0.05) 1,368 (±327) 1051 (±279)
(his)hGH(4) 680 (±167) 0.61 (±0.05) 1,033 (±92) 1.30 (±0.17) 974 (±84) 853 (±91)
30KPEG-pAF35(his)hGH(4) 1,885 (±1,011) 4.85 (+0.80) 39,918 (±22,683) 19.16 (±4.00) 35 (±27) 268 (±236)
30KPEG-pAF92(his)hGH(6) 663 (±277) 4.51 (+0.90) 10,539 (±6,639) 15.05 (±2.07) 135 (±90) 959 (±833)
30KPEG-pAF131(his)hGH (5) 497 (±187) 4.41 (±0.27) 6,978 (±2,573) 14.28 (±0.92) 161 (±61) 1,039 (±449)
30KPEG-pAF134(his)hGH (3) 566 (±204) 4.36 (±0.33) 7,304 (±2,494) 12.15 (±1.03) 151 (±63) 931 (±310)
30KPEG-pAF143(his)hGH (5) 803 (±149) 6.02 (±1-43) 17,494 (±3,654) 18.83 (±1.59) 59 (±11) 526 (±213)
30KPEG-pAF145(his)hGH (5) 634 (+256) 5.87 (+0.09) 13,162 (±6,726) 17.82 (+0.56) 88 (±29) 743 (+252)
Drug efficacy study
Obtain the male Sprague-Dawley rat hypophysectomize from Charles River Laboratories.Hypophysectomize with the surgery modus operandi when big in 3 to 4 weeks.Allow animal to adapt to the period in 3 weeks, monitor body weight during this period.The animal that has 0 to 8g weight increase in the research preceding 7 day period of beginning is included in wherein, and divides at random to the treatment group.Bolus dosage or per daily dose are given in throwing to subcutaneous rat.During whole research, in order rat weighed every day, benumb, blood drawing and administration (when suitable).Use is taken a blood sample from the socket of the eye hole through the kapillary of heparinization, and blood sample is placed the micro-centrifuge tube that applies through EDTA.Preserve until analysis down by the centrifugation separated plasma and at-80 ℃.
Figure 17 be presented in the rat body that hypophysectomizes behind the single subcutaneous administration average (+/-S.D.) plasma concentration.(every group 5 to 7 of n=) provides the proteinic single bolus of 2.1mg/kg dosage to rat.Show to comprise on each position in comfortable two different positionss (position 35,92) result of covalently bound alpha-non-natural amino acid to 30kDa PEG to the hGH polypeptide of acetylphenylalanine.According to the indicated timed interval and collection plasma sample, and examine and determine described injection compound.
Peptide IGF-1 is the family member of somatomedin or insulin-like growth factor.The multiple growth enhancing effect of IGF-1 mediating growth hormone.Use competition desmoenzyme immunoassays cover to organize at the rat/mouse IGF-1 standard substance that is provided (Diagnosic Systems Laboratories) and measure IGF-1 concentration.By t test use two tails distribute (not in pairs, etc. variance) measure significant difference.Figure 18, A figure shows the assessment of compound in the rat body that hypophysectomizes.To subcutaneous single dose or the per daily dose of providing of rat (5 to 7 every group of n=).Every day in regular turn with animal weigh, anaesthetize, blood drawing and administration (when suitable).Demonstration is for placebo treatment, wild-type hGH (hGH), through the hGH of His mark ((his) hGH) with comprise the covalently bound body weight result to the hGH polypeptide treatment of acetylphenylalanine to 30kDa PEG on position 35 and 92.Figure 18, B figure---be presented at comprise non-naturally encoded amino acid whose PEGization hGH polypeptide single dose and offer medicine after, for the figure of the influence of circulating plasma IGF-1 level.Lines are represented standard deviation.At Figure 18, among the A figure, 30KPEG-pAF35 (his) hGH compound is different from (p＜0.0005) 30KPEG-pAF92 (his) hGH compound (observing bigger weight increase therein) at the 9th day weight increase on statistics.
Figure 18, C figure shows the assessment of compound in the rat body that hypophysectomizes.To subcutaneous bolus dosage or the per daily dose of providing of rat (every group 11 of n=).In order animal weighed every day, anaesthetize, blood drawing and administration (when suitable).For placebo treatment, wild-type hGH (hGH) with on position 92,134,145,131 and 143, comprise covalently bound alpha-non-natural amino acid to 30kDa PEG the hGH polypeptide of acetylphenylalanine is shown the body weight result.Figure 18, figure D---pictorialization compared to placebo treatment and wild-type hGH, the influence in the PEGization hGH polypeptide single dose dispensing back that comprises non-naturally encoded amino acid (position 92,134,145,131,143) for circulating plasma IGF-1 level.Figure 18, figure E show corresponding to the PEGization hGH polypeptide that comprises non-naturally encoded amino acid (position 92,134,145,131,143) on average (+/-S.D.) plasma concentration.According to the indicated timed interval and collection plasma sample, and examine and determine described injection compound.Lines are represented standard deviation.
Example 31
Comprise the security of non-naturally encoded amino acid whose PEGization hGH and/or the human clinical trial of usefulness.
PurposeTo subcutaneous throwing give comprise non-naturally encoded amino acid whose PEGization recombinant human hGH and one or more commercially available hGH products (include, but is not limited to Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono)) security and pharmacokinetics compare.
The patient With 18 ages deposit 20 and 40 years old between, body weight deposit 60 and 90kg between the healthy volunteer add in the research.The person under inspection will be without any significant abnormal experimental value clinically for blood or blood plasma chemistry and the screening of urine toxicology, HIV screening and viral hepatitis type b surface antigen.It should not have following any sign: hypertension; The hemopathic medical history of any primary; Remarkable hepatopathy, ephrosis, cardiovascular diseases, gastrointestinal disorder, urogenital disease, metabolic disease, neuropathic medical history; The medical history of anemia or epilepsy (seizure disorder); Known sensitivity for bacterium or Mammals derived products, PEG or human serum albumin; The habitual beverage that contains caffeine of drinking in a large number; Participate in any other clinical trial or once transfused blood or donated blood in 30 days beginning one's study; Once be exposed to hGH in 3 months beginning one's study; Once begin one's study ill in 7 days; And has remarkable abnormality for the health check-up before the research or clinical labororatory's assessment of beginning one's study in 14 days.All persons under inspection are appreciable for security, and gather the whole blood sampling that is used for the pharmacokinetics analysis as scheduled.All researchs are all agreed to carry out according to institutional ethics committee approval and patient.
Research and designThis will be for for the Phase I of healthy male volunteers, single center, open-label, random packet, two-stage crossing research.18 persons under inspection are assigned as two treatment sequence set (9 person under inspection/groups) randomly.Use isodose comprise non-naturally encoded amino acid whose PEGization hGH and selected commercially available prod, through two independently the administration stage with GH as bolus in the subcutaneous injection of thigh top.The dispensing dosage and the frequency of commercially available prod should be indicated as packaging label.Can will use the extra administration, administration frequency of commercially available prod or other to want parameter to be added in the research by comprising extra person under inspection group.(washout) phase of eliminating by 14 days is separated each administration stage.In before per two administration stages 12 hours and afterwards 72 hours the person under inspection is limited in the research centre, but between the administration stage, does not limit.If also exist extra administration, frequency or other parameter to need test, can add extra person under inspection's group so for PEGization hGH.Can be used for this research through approval for the human multiple GH prescription that uses.Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono) be to be the human commercially available GH product that uses through approval.The experimental formula of hGH is to comprise non-naturally encoded amino acid whose PEGization hGH.
Blood samplingGive the direct venipuncture of hGH front and back draws blood continuously by throwing.Following whenabouts after about before 30,20 and 10 minutes of administration (3 baseline sample) and administration obtains to be used to measure the venous samples can (5mL) of Serum GH concentration: 30 minutes and 1,2,5,8,12,15,18,24,30,36,48,60 and 72 hour.Each serum sample is divided into two parts of aliquots containigs.All serum samples are all-20 ℃ of storages down.On dry ice, load serum sample.(immediately) and the 19th day morning are carried out empty stomach clinical labororatory test (hematology, serum chemistry character and urinalysis) before (immediately), the 4th day morning, the 16th day administration before the 1st day the initial administration.
Bioanalytical methodELISA cover group program (Diagnostic Svstems Laboratory[DSL], Webster TX) is used to measure Serum GH concentration.
Security is measured6,24,48 and 72 hour record sign of life after each preceding and each administration of administration (the 1st day and the 16th day).Security measure to be with the incidence of adverse events and type and the clinical labororatory's test foundation that is changed to compared to baseline.In addition, the variation of measuring in (comprising blood pressure) and the physical examination result at sign of life before research is assessed.
Data analysisBy deducting average baselining GH concentration (measuring) with the value after each administration, the serum-concentration value after the administration is proofreaied and correct baseline GH concentration before the administration by the GH content of three duplicate samples of gathering in 30,20 and 10 minutes before the self administration of medication is averaged.If Serum GH concentration is lower than the quantification content of calibrating before the administration, it is not included in the calculating of mean value so.By the serum-concentration data determination pharmacokinetic parameter of proofreading and correct the baseline GH concentration before the administration.On Digital Equipment Corporation VAX 8600 computer systems, use the computed in software pharmacokinetic parameter of the BIOAVL of latest edition by the model independent solution.Measure following pharmacokinetic parameter: peak serum concentration (C Max); Time (t to peak serum concentration Max); Utilize that trapezoidal integration rule calculates from 0 blood sampling time (AUC to the end 0-72) concentration-time curve under area; Eliminate transformation period (t with end via the elimination factor constant calculations 1/2).The elimination factor constant is estimated in linear regression by consecutive numbers strong point in the terminal linearity region of logarithm-linear concentration-time curve.Calculate mean value, standard deviation (SD) and the variation coefficient (CV) of pharmacokinetic parameter for each treatment.The ratio of calculating parameter mean value (anticorrosion prescription/non-anticorrosion prescription).
The incidence of safety results adverse events is evenly distributed in the treatment group.There is not the noticeable change clinically of before baseline or research clinical labororatory test or blood pressure, and do not have before the research considerable change of measuring about physical examination result and sign of life.The security overview of two treatment groups should be expressed as similar.
Pharmacokinetics resultDeposit one or more commercially available hGH products of on each measured time point all 18 persons under inspection being taken single dose and (include, but is not limited to Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono)) the average serum GH concentration-time overview after (not check baseline GH content) with comprise non-naturally encoded amino acid whose PEGization hGH and compare.All persons under inspection should have the preceding baseline GH concentration of administration in the normal physiological scope.By the serum data determination pharmacokinetic parameter of proofreading and correct average baselining GH concentration before the administration, and measure C MaxAnd t MaxSelected clinical comparer (Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono)) average t MaxSignificantly be shorter than the average t that comprises non-naturally encoded amino acid whose PEGization hGH MaxCompared to the terminal transformation period that comprises non-naturally encoded amino acid whose PEGization hGH, the terminal elimination half life values of commercially available hGH product is significantly shorter.
Although this research is carried out, in other patient colony, also can expect similar absorption feature and security overview in the healthy male person under inspection; Such as the sex patient who suffers from cancer or chronic renal failure, paediatrics patients with renal failure, patient or the predetermined patient who carries out elective surgery in body storage type program.In a word, the single dose that gives through subcutaneous throwing comprises that non-naturally encoded amino acid whose PEGization hGH will be for safety, and the healthy male person under inspection has good drug-resistant power to it.According to incidence, clinical labororatory's value, sign of life and the physical examination result of comparative adverse events, the hGH of commercial form will equate with the security overview that comprises non-naturally encoded amino acid whose PEGization hGH.Comprise non-naturally encoded amino acid whose PEGization hGH and provide big clinical efficacy for patient and healthcare provider potentially.
Example 32
In following example, aHGH and PEG- aHGH is respectively hGH and has PEGization hGH to the SEQ ID NO:2 of acetylphenylalanine on position 35, and PEG is by to connect the formed oxime key of acetylphenylalanine in described PEGization hGH, and wherein PEG is linear 30kDa PEG.
The calibrating of STAT5 phosphorylation
The interaction of hGH and its acceptor causes signal transducer and transcribes the tyrosine phosphorylation of family member's activator (STAT5) in human IM-9 lymphocyte series.By cumulative with concentration aHGH and PEG- aHGH activation IM-9 cell then to phosphorus-STAT5 ICF immunity inspection method dyeing, thereby is measured aHGH and PEG- aThe concentration of hGH (requires 50% maximum STAT5 phosphorylation (EC 50)).According to the WHO of the World Health Organization) EC of standard hGH 50As 1.0, measure aHGH is compared to the relative EC of WHO hGH 50Be 1.1, and PEG- aThe relative EC of hGH 50Be 10.9.
Example 33:
The propagation calibrating
Proliferating cells ties up to and in vitro measures PEG-by means of response tethelin aThe hGH activity.An example of described clone is the rat GHR[L43R that is called 2E2-2B12-F4]/the BAF3 cell clone, it is the clone by using rat GHR (replacement that has Leu to Arg on position 43) stable transfection mouse BAF3 cell to be produced.The conversion of Leu-43 to Arg-43 makes rat GHR more " similar to the mankind " in the rat receptor, and therefore realizes effective hGH combination.Adopt activation to separate rat GHR[L43R]/bromodeoxyribouridine (BrdU) mark of BAF3 cell provides a kind of inducing growth hormone that is used for to breed quantitative high separation, radiationless property method.EC with WHO hGH 50Be set at unanimity, measure aHGH and PEG- aHGH is compared to the EC of WHO hGH 50Be respectively 1.06 ± 0.29 (n=11) and 5.37 ± 1.23 (n=9).
Example 34.
The effect research of PEGization hGH
In the rat model of growth hormone deficiency, carry out preclinical effect research.In described model, when about 4 weeks are big with the surgery modus operandi gland that hypophysectomizes.Monitor the body weight of these rats that hypophysectomize after surgery, will show in the preceding 7 day period of research beginning that animal less than the 7.5g weight increase is considered as that success hypophysectomizes, and it is randomized into the treatment group.Unsound with showing that the animal that loses weight greater than 2.5g is considered as, and get rid of outside research.The described research of 3 weeks beginning approximately after surgery.
Weekly with placebo or cumulative PEG- aHGH dosage is to animals administer (that is, the 0th day and administration in the 7th day).An additional procedures group is taken strong person of outstanding talent peaceful (genotropin) or placebo every day.Every day before administration to body weight and blood sampling.All animals were death in the 14th day.Figure 28 shows the blood plasma IGF-1 level during the whole research.Observing IGF-1 firm, that decide on dosage increases.
Behind each dosing interval, observe the increase that IGF-1 Cmax decides on dosage.Since the fitting of a curve of the cmax value of the administration first time minimum (Eo) and maximum (Emax) IGF-1 Cmax are estimated as 136.7 and 1 respectively, 136.2ng/mL, and measure ED 50Be by 0.136mg/kg (table 8).
Blood plasma IGF-1 level is also shown in the increase of significantly deciding on dosage when being expressed as AUC.For this parameter, AUC is expressed as the AUC that is higher than effective IGF-1 blood plasma level.Previous work shows, the lasting IGF-1 concentration of the above 34ng/mL of the baseline weight increase of rat that causes hypophysectomizing.In current research, running through the baseline IGF-1 level that described research measures from placebo is 128ng/mL.The increment of the above 34ng/mL of 128ng/mL is estimated as effective IGF-1 level of 162ng/mL.Therefore the above AUC of 162ng/mL is complete.Analyze by this, the AUC that decides on dosage that observes behind each dosing interval more than level of significance increases.Since the fitting of a curve of the AUC value of the administration first time Eo and Emax IGF-1 AUC are estimated as 0 and 5 respectively, 029.8ngXhr/mL, and measure the ED of described parameter 50Be by 0.909mg/kg (table 8).
Measure the IGF-1 inducing sustained time of level more than the estimation level of significance after the administration first time.Under higher dosage, the IGF-1 level is not returned baseline before the administration second time.In these cases, use terminal slope as calculated that IGF-1 concentration is extrapolated to level of significance.By this estimation, the increase that the time length of IGF-1 concentration more than level of significance decides on dosage is conspicuous.Fitting of a curve is estimated as 0 and 8.84 day respectively with minimum and maximum time length, and measures the ED of described parameter 50Be by O.173mg/kg (table 8).
Osteogenesis is measured and calculated PEG-as extra drug effect aHGH ED 50When research finishes, gather shin bone, and immersion liquid is fixed in 10% formalin through neutral buffered (formalin), carries out radiograph then from each animal.Display result in Figure 29.Through PEG- aThere is the increase of significantly deciding on dosage in the shin bone length of the animal of hGH treatment.In addition, exist dosage to save effect compared to every day through the animal of the peaceful treatment of strong person of outstanding talent.Throw the PEG-that the peaceful amount of strong person of outstanding talent of giving equals weekly the 2.1mg/kg that (that is, the 0th day and the 7th day) provide through 7 days the timed interval aHGH.PEG-for 0.42 to 1.0mg/kg aHGH shows and inducing that the peaceful similarly shin bone length of strong person of outstanding talent increases.This is for PEG- aThe hGH expression is rather saved effect more than or equal to 50% dosage compared to strong person of outstanding talent.
PEG- aHGH also inductor payes attention to dosage and fixed increase (Figure 30).In addition, induce PEG-as what shin bone length increased aIt is conspicuous that hGH is better than the peaceful dosage saving effect of strong person of outstanding talent.The peaceful treatment of using 0.3mg/kg/ days every day of strong person of outstanding talent is induced and is originated in the 0th day body weight change at the 14th day 27.74 (+/-7.92) %.To throw the PEG-that gives with the GH equal quantities aHGH (being that 2.1mg/kg was at the 0th day and the 7th day) induces the peaceful bigger weight increase compared to strong person of outstanding talent.The PEG-of this dosage aHGH causes originating in the 0th day body weight change at the 14th day 33.66 (+/-7.55) %.By throwing the PEG-that gives at the 0th day and the 7th day than low dosage with 1.0mg/kg aHGH induces body weight per-cent to change the body weight change of inducing at the 14th day 27.02 (+/-3.97) %.This causes for PEG- aThe dosage of hGH approximate 50% is saved effect.The non-PEGization GH dispensing of 2.1mg/kg dosage can't be induced weight increase weekly.
The fitting of a curve of body weight per-cent variation in the 14th day changes minimum and weight limit per-cent (by PEG- aHGH induces) be estimated as 7.17 and 50.65 respectively.By this curve, measure the ED of described parameter 50Be by 1.097mg/kg (table 8).
Table 8. uses PEG- aThe effect research result of hGH
Pharmacodynamics parameter (effect) The treatment instructions about how to take medicine Effect scope (Eo to Emax) ED 50(mg/kg)
IGF-1 induces: IGF-1?Cmax Behind the single subcutaneous administration 136.7 to 1,136.2 mg/mL 0.136
The IGF-1 AUG that level of significance is above Behind the single subcutaneous administration 0 to 5,029.8 ngXhr/mL 0.909
The IGF-1 time length that level of significance is above Behind the single subcutaneous administration 0 to 8.84 day 0.173
Shin bone length The 14th day, 2 times weekly behind the subcutaneous administration 30.52 to 33.67 mm 0.424
Body weight per-cent changes The 14th day, 2 times weekly behind the subcutaneous administration 7.17 to 50.65% 1.097
Also be used to examine and determine PEG-for gathering with the identical plasma sample that is used for the IGF-1 concentration determination aHGH concentration.Used ELISA calibrating is exclusively used in hGH, and it is for PEG- aHGH shows lower but still firm response, and is not used in detection rat GH.In Figure 31, show PEG- aHGH plasma concentration-time diagram.The PEG-that decides on dosage aHGHCmax increases.PEG- aTime length and the total PEG-of hGH in circulation aHGH AUC increases in the mode of deciding on dosage.Throw the strong person of outstanding talent who gives and rather can not detect in blood plasma every day, may be because remove fast.PEG- aHGH exposes as a result the usefulness of deciding on dosage shown in the support above.
The research of the above-mentioned rat that hypophysectomizes has shown following content:
1.PEG- aHGH increases the IGF-1 plasma concentration on dosage with deciding.IGF-1 Cmax that level of significance is above and IGF-1 AUC and time length all increase on dosage with deciding.
2. shin bone length and body weight increase on dosage with deciding.
3. explanation is thrown the strong person of outstanding talent give rather more than or equal to 50% dosage saving compared to every day.
4.PEG- aHGH shows the increase that Cmax, AUC and persistence are decided on dosage in circulation, it is relevant with above drug effect effect.
Example 35
Pharmacokinetics research
The rat pharmacokinetics
In example 34 above, show and in the rat disease model, throw the PEG-that gives with various dosage aThe plasma concentration of hGH is with respect to the overview of time.
The primates pharmacokinetics
The assessment difference only is that molecule contains the PEG-of methionine(Met) on the N end position in macaque aHGH type (PEG- a(met) pharmacokinetics character (Figure 32) hGH).
Throw with the subcutaneous or intravenous dosages of 0.75mg/kg or 0.15mg/kg respectively and give PEG- a(met) single injection of hGH.Compared to having PEG- aIn the rat of hGH 7.05 (+/-0.47) hour, the apparent terminal transformation period is 12.23 (+/-1.72) hour.The transformation period of intravenously dispensing is 6.42 (+/-0.51) hour.Be respectively 362,443 and 312,440ngXhrXkg/mL/mg through the gauged AUC value of dosage for subcutaneous and intravenous administration.The biological activity of calculating by subcutaneous administration is 116%.
Example 36
The ultimate principle of proposed dosage and administration instructions about how to take medicine in clinical protocol
Determine the optimum instructions about how to take medicine of PEGization GH in human body by the research that is listed in down in the animal.
The dosage saving that the rat effect research is observed in the effect research before clinical one by one (promptly with strong person of outstanding talent would rather than usefulness, but have less PEG- aHGH dosage) be used for the required dosage estimation of human body biological activity.
Pharmacokinetics assessment in rat and macaque---the use allometron is estimated the pharmacokinetic parameter in the human body.
The acute safety research of single dose in rat and macaque is used for the assessment exposure and measures NOAEL (not observing the ill effect level).
A month repeated doses GLP of rat safety research---be higher than 2 times, 6 times of the human dosage of maximum recommended (MRHD) and 20 multiple doses are assessed security after one month being exposed to.For paediatrics GHD, MRHD=0.36mg/kg/ week.
A month repeated doses GLP of macaque safety research---be higher than 2 times, 10 times of the human dosage of maximum recommended (MRHD) and 30 multiple doses are assessed security after one month being exposed to.For paediatrics GHD, MRHD=0.36mg/kg/ week.
Six months repeated doses GLP of rat safety research.Exposed six months so that assess security behind 1 to 2 times and 10 times of the exposure that exposing as in the rat observed under the MRHD among the mankind in administration.
Six months repeated doses GLP of macaque safety research.Exposed six months so that assess security behind 1 to 2 times and 10 times of the exposure that exposing as in the macaque observed under the MRHD among the mankind in administration.
Should be appreciated that, described herein example and embodiment are only for purposes of illustration, and will be easy to the those skilled in the art expects according to its various modifications made or variation, and will be included in the category of the spirit and scope of the application's case and the claim of enclosing.The open case of all that quote in the application's case, patent, patent application case and/or other document are all to be incorporated herein by reference for the purpose of all purposes, and it quotes degree just as individually each open case, patent, patent application case and/or other document being incorporated into by reference for the purpose of all purposes.
Table 9: the sequence of being quoted.
SEQ ID# The sequence title
1 The full length amino acid sequence of hGH
2 The mature amino acid sequence of hGH (with merit abnormal shape 1)
3 Wherein lack the 20-kDa hGH varient of hGH residue 32 to 46
21 The nucleotide sequence of total length hGH
22 The nucleotide sequence of ripe hGH
Sequence table
<110>Cho，Ho?S
Daniel，Thomas?0.
DiMarchi，Richard?D.
Hays，Anna-Maria
Wilson，Troy?E.
Sim，Bee-Cheng
Litzinger, David C.＜120〉modified human growth hormone＜130〉AMBX-0076.00PCT
<150>60/638,616
<151>2004-12-22
<150>60/727,996
<151>2005-10-17
<160>22
<170>PatentIn?version?3.3
<210>1
<211>217
<212>PRT
＜213〉homo sapiens
<400>1
Met?Ala?Thr?Gly?Ser?Arg?Thr?Ser?Leu?Leu?Leu?Ala?Phe?Gly?Leu?Leu
5 10 15
Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser?Ala?Phe?Pro?Thr?Ile?Pro?Leu
20 25 30
Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln
35 40 45
Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys
50 55 60
Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe
65 70 75 80
Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys
85 90 95
Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp
100 105 110
Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val
115 120 125
Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu
130 135 140
Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg
145 150 155 160
Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser
165 170 175
His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe
180 185 190
Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys
195 200 205
Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
210 215
<210>2
<211>191
<212>PRT
＜213〉homo sapiens
<400>2
Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg
1 5 10 15
Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu
20 25 30
Glu?Ala?Tyr?Ile?Pro?Lys?Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro
35 40 45
Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg
50 55 60
Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu
65 70 75 80
Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val
85 90 95
Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp
100 105 110
Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu
115 120 125
Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser
130 135 140
Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr
145 150 155 160
Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe
165 170 175
Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
180 185 190
<210>3
<211>176
<212>PRT
＜213〉homo sapiens
<400>3
Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg
1 5 10 15
Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Asn
20 25 30
Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn
35 40 45
Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser
50 55 60
Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser
65 70 75 80
Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr
85 90 95
Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg
100 105 110
Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr
115 120 125
Ser?Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn
130 135 140
Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr
145 150 155 160
Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
165 170 175
<210>4
<211>77
<212>DNA
＜213〉Methanococcus jannaschii
<400>4
ccggcggtag?ttcagcaggg?cagaacggcg?gactctaaat?ccgcatggcg?ctggttcaaa 60
tccggcccgc?cggacca 77
<210>5
<211>88
<212>DNA
＜213〉have a liking for the salt bacillus specie
<400>5
cccagggtag?ccaagctcgg?ccaacggcga?cggactctaa?atccgttctc?gtaggagttc 60
gagggttcga?atcccttccc?tgggacca 88
<210>6
<211>89
<212>DNA
＜213〉have a liking for the salt bacillus specie
<400>6
gcgagggtag?ccaagctcgg?ccaacggcga?cggacttcct?aatccgttct?cgtaggagtt 60
cgagggttcg?aatccctccc?ctcgcacca 89
<210>7
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>7
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?Asm?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>8
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>8
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>9
<211>305
<212>PRT
＜213〉Methanococcus jannaschii
<400>9
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>10
<211>305
<212>PRT
＜213〉Methanococcus jannaschii
<400>10
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>11
<211>305
<212>PRT
＜213〉Methanococcus jannaschii
<400>11
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>12
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>12
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?Ash?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>13
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>13
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>14
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>14
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>15
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>15
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>16
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<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?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>17
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>17
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?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys
290 295 300
Arg?Leu
305
<210>18
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>18
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>19
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<400>19
Met?Asp?Glu?Phe?Glu?MetIle?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>20
<211>306
<212>PRT
＜213〉Methanococcus jannaschii
<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>654
<212>DNA
＜213〉homo sapiens
<400>21
atggctacag?gctcccggac?gtccctgctc?ctggcttttg?gcctgctctg?cctgccctgg 60
cttcaagagg?gcagtgcctt?cccaaccatt?cccttatcca?ggctttttga?caacgctatg 120
ctccgcgccc?atcgtctgca?ccagctggcc?tttgacacct?accaggagtt?tgaagaagcc 180
tatatcccaa?aggaacagaa?gtattcattc?ctgcagaacc?cccagacctc?cctctgtttc 240
tcagagtcta?ttccgacacc?ctccaacagg?gaggaaacac?aacagaaatc?caacctagag 300
ctgctccgca?tctccctgct?gctcatccag?tcgtggctgg?agcccgtgca?gttcctcagg 360
agtgtcttcg?ccaacagcct?ggtgtacggc?gcctctgaca?gcaacgtcta?tgacctccta 420
aaggacctag?aggaaggcat?ccaaacgctg?atggggaggc?tggaagatgg?cagcccccgg 480
actgggcaga?tcttcaagca?gacctacagc?aagttcgaca?caaactcaca?caacgatgac 540
gcactactca?agaactacgg?gctgctctac?tgcttcagga?aggacatgga?caaggtcgag 600
acattcctgc?gcatcgtgca?gtgccgctct?gtggagggca?gctgtggctt?ctag 654
<210>22
<211>576
<212>DNA
＜213〉homo sapiens
<400>22
ttcccaacca?ttcccttatc?caggcttttt?gacaacgcta?tgctccgcgc?ccatcgtctg 60
caccagctgg?cctttgacac?ctaccaggag?tttgaagaag?cctatatccc?aaaggaacag 120
aagtattcat?tcctgcagaa?cccccagacc?tccctctgtt?tctcagagtc?tattccgaca 180
ccctccaaca?gggaggaaac?acaacagaaa?tccaacctag?agctgctccg?catctccctg 240
ctgctcatcc?agtcgtggct?ggagcccgtg?cagttcctca?ggagtgtctt?cgccaacagc 300
ctggtgtacg?gcgcctctga?cagcaacgtc?tatgacctcc?taaaggacct?agaggaaggc 360
atccaaacgc?tgatggggag?gctggaagat?ggcagccccc?ggactgggca?gatcttcaag 420
cagacctaca?gcaagttcga?cacaaactca?cacaacgatg?acgcactact?caagaactac 480
gggctgctct?actgcttcag?gaaggacatg?gacaaggtcg?agacattcct?gcgcatcgtg 540
cagtgccgct?ctgtggaggg?cagctgtggc?ttctag 576

Claims (79)

1. hormonal composition, it comprises the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.
2. hormonal composition according to claim 1, wherein said GH are human growth hormone (hGH).
3. hormonal composition according to claim 2, wherein said hGH comprise at least about 80% with the identical sequence of SEQ ID NO:2.
4. hormonal composition according to claim 2, wherein said hGH comprises the sequence of SEQ ID NO:2.
5. hormonal composition according to claim 1 and 2, wherein said GH comprise non-naturally encoded amino acid (NEAA).
6. hormonal composition according to claim 5, it is included in the oxime key between described NEAA and the described water-soluble polymers.
7. hormonal composition according to claim 6, wherein said NEAA comprises carbonyl.
8. hormonal composition according to claim 7, wherein said NEAA comprises ketone group.
9. hormonal composition according to claim 8, wherein said NEAA are to acetylphenylalanine.
10. hormonal composition according to claim 9 wherein replaces acetylphenylalanine with described in the position corresponding with the position 35 of SEQ ID NO:2.
11. hormonal composition according to claim 1 and 2, wherein said water-soluble polymers comprise polyoxyethylene glycol (PEG).
12. hormonal composition according to claim 11, wherein said PEG are linear PEG.
13. hormonal composition according to claim 12, wherein said PEG have the molecular weight between about 0.1kDa and the about 100kDa.
14. hormonal composition according to claim 12, wherein said PEG have the molecular weight between about 1kDa and the about 60kDa.
15. hormonal composition according to claim 12, wherein said PEG have the molecular weight between about 20kDa and the about 40kDa.
16. hormonal composition according to claim 12, wherein said PEG has the molecular weight of about 30kDa.
17. hormonal composition according to claim 11, wherein said PEG are branch PEG.
18. hormonal composition according to claim 17, wherein said PEG have the molecular weight between about 1kDa and the about 100kDa.
19. hormonal composition according to claim 17, wherein said PEG have the molecular weight between about 30kDa and the about 50kDa.
20. hormonal composition according to claim 17, wherein said PEG has the molecular weight of about 40kDa.
21. hormonal composition according to claim 7, wherein said water-soluble polymers comprises PEG.
22. hormonal composition according to claim 21, wherein said PEG are linear PEG.
23. hormonal composition according to claim 21, wherein said PEG have the molecular weight between about 0.1kDa and the about 100kDa.
24. hormonal composition according to claim 21, wherein said PEG have the molecular weight between about 1kDa and the about 60kDa.
25. hormonal composition according to claim 21, wherein said PEG have the molecular weight between about 20kDa and the about 40kDa.
26. hormonal composition according to claim 21, wherein said PEG has the molecular weight of about 30kDa.
27. hormonal composition according to claim 7, wherein said PEG are branch PEG.
28. hormonal composition according to claim 28, wherein PEG has the molecular weight between about 1kDa and the about 100kDa.
29. hormonal composition according to claim 28, wherein said PEG have the molecular weight between about 30kDa and the about 50kDa.
30. hormonal composition according to claim 28, wherein said PEG has the molecular weight of about 40kDa.
31. hormonal composition according to claim 10, wherein said water-soluble polymers are PEG.
32. hormonal composition according to claim 31, wherein said PEG are linear PEG.
33. hormonal composition according to claim 32, wherein said PEG have the molecular weight between about 0.1kDa and the about 100kDa.
34. hormonal composition according to claim 32, wherein said PEG have the molecular weight between about 1kDa and the about 60kDa.
35. hormonal composition according to claim 32, wherein said PEG have the molecular weight between about 20kDa and the about 40kDa.
36. hormonal composition according to claim 32, wherein said PEG has the molecular weight of about 30kDa.
37. hormonal composition according to claim 32, wherein said GH are hGH.
38. according to the described hormonal composition of claim 37, wherein said hGH comprises SEQ ID NO:2.
39. hormonal composition according to claim 1, it comprises the GH that is connected with a plurality of water-soluble polymerss by a plurality of covalent linkage, and at least one in the wherein said covalent linkage is the oxime key.
40. according to the described hormonal composition of claim 39, wherein said GH is human growth hormone (hGH).
41. according to the described hormonal composition of claim 40, wherein said hGH comprises at least about 80% sequence identical with SEQ IDNO:2.
42. hormonal composition according to claim 2, wherein said hGH comprises the sequence of SEQ ID NO:2.
43. according to claim 39 or 41 described hormonal compositions, wherein said GH comprises a plurality of NEAA.
44. hormonal composition, it comprises the hGH that is connected by the linear PEG of oxime key and at least one, and wherein said hGH comprises the aminoacid sequence of SEQ ID NO:2 and comprises the NEAA that at least one replaces in one or more positions that are selected from the group that is made up of following each residue position: residue 1-5,6-33,34-74,75-96,97-105,106-129,130-153,154-183 and 184-191.
45. according to the described hormonal composition of claim 44, wherein replace with described NEAA in one or more positions that are selected from the group that forms by following each residue position: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191 and 192 (that is, at proteinic carboxyl terminales).
46., wherein replace with described NEAA: position 35,92,131,134,143 and 145 in one or more positions that are selected from the group that forms by following each residue position according to the described hormonal composition of claim 44.
47., wherein replace with described NEAA: position 30,35,74,92,103,143 and 145 in one or more positions that are selected from the group that forms by following each residue position according to the described hormonal composition of claim 44.
48., wherein replace with described NEAA: position 35,92,143 and 145 in one or more positions that are selected from the group that forms by following each residue position according to the described hormonal composition of claim 44.
49. according to the described hormonal composition of claim 44, it is included in the NEAA that replaces on the position 35.
50. according to claim 44,45,46,47,48 or 49 described hormonal compositions, it is included as the NEAA to acetylphenylalanine.
51. according to the described hormonal composition of claim 50, wherein said PEG has the molecular weight between about 0.1kDa and the about 100kDa.
52. according to the described hormonal composition of claim 50, wherein said PEG has the molecular weight between about 1kDa and the about 60kDa.
53. according to the described hormonal composition of claim 50, wherein said PEG has the molecular weight between about 20kDa and the about 40kDa.
54. according to the described hormonal composition of claim 50, wherein said linear PEG has the molecular weight of about 30kDa.
55. the method for the GH that a manufacturing is connected with water-soluble polymers by the oxime key, it comprises makes the GH that comprises the NEAA that contains carbonyl contact with the PEG oxyamine being suitable for forming under the condition of oxime key.
56. according to the described method of claim 55, wherein said NEAA contains ketone group.
57. according to the described method of claim 56, wherein said NEAA is to acetylphenylalanine.
58., wherein (for example, acetylphenylalanine is replaced with described corresponding to the position of the amino acid among the SEQ IDNO:2 35 in hGH) at described GH according to the described method of claim 57.
59. according to the described method of claim 55, wherein said PEG oxyamine is mono methoxy PEG (MPEG) oxyamine.
60. according to the described method of claim 59, wherein said MPEG oxyamine is linear.
61. according to the described method of claim 60, the molecular weight of wherein said PEG is about 20 to 40kDa.
62. according to the described method of claim 61, the molecular weight of wherein said PEG is about 30kDa.
63. according to the described method of claim 62, wherein said MPEG oxyamine is mono methoxy-PEG-2-amino oxygen base ethamine carbamate hydrochloride of linear 30kDa.
64. according to the described method of claim 55, it further comprises makes the GH that comprises NEAA by the following method, described method comprises: will
(i) coding GH nucleic acid (wherein said nucleic acid has been modified so that for incorporating into of described NEAA providing signal) and
(ii) described NEAA introduces in the organism, and wherein said organism can be incorporated described NEAA in the protein in response to the described signal of described (i) nucleic acid.
65. according to the described method of claim 55, wherein said condition comprises:
(i) described GH is mixed with MPEG to produce the MPEG-GH mixture, wherein the ratio of MPEG: GH is about 5 to 10,
(ii) the pH value is about 4 to 6; With
(iii) at room temperature with the soft stir about of described MPEG-GH mixture 10 to 50 hours.
66. according to the described method of claim 55, it further comprises the described GH of purifying.
67. according to the described method of claim 56, wherein the GH that obtains by described method is at least about 99% pure.
68. a medical composition, it comprises:
(i) hormonal composition, it comprises the tethelin that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key; With
(ii) pharmaceutically acceptable vehicle.
69. according to the described medical composition of claim 68, wherein said GH is hGH.
70. according to claim 68 or 69 described medical compositions, wherein said composition comprises pharmaceutically acceptable injection prescription liquid.
71. according to the described medical composition of claim 69, wherein said GH comprises NEAA.
72. according to the described medical composition of claim 71, wherein said water-soluble polymers comprises PEG.
73. according to the described medical composition of claim 71, wherein said PEG is linear PEG.
74. according to the described medical composition of claim 73, wherein said PEG is the linear PEG of about 30kDa, and described GH is corresponding to the position of the amino acid 35 among the SEQ ID NO:2 hGH through acetylphenylalanine is replaced, and wherein described to acetylphenylalanine and described PEG between the described oxime key of formation.
75. a methods of treatment, it comprises throws the individuality that gives the needs treatment with the hormonal composition of significant quantity, and described hormonal composition comprises the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.
76. according to the described method of claim 75, wherein said individuality suffers from the illness that is selected from the group that is made up of following illness: the paediatrics growth hormone deficiency, special send out property of short and small stature, the Childhood outbreak grownup's growth hormone deficiency, the grownup's growth hormone deficiency or the Secondary cases growth hormone deficiency of Adulthood outbreak.
77. methods of treatment, it comprises throws the individuality that gives the needs treatment with the hormonal composition of significant quantity, described hormonal composition comprises the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, wherein said water-soluble polymers is a linear polymer, and wherein with weekly only once, whenever biweekly or mensal frequency throw and to give described hormonal composition.
78. a hormonal composition, it comprises GH, wherein has average serum transformation period at least about 12 hours as described GH when Mammals is given in subcutaneous throwing.
79. a hormonal composition, it comprises the GH that is connected with PEG by the oxime key, wherein when the average serum transformation period of described GH when Mammals is given in subcutaneous throwing be comprise no described PEG GH composition serum half-life at least about 7 times.
CNA2005800444641A 2004-12-22 2005-12-21 Modified human growth hormone Pending CN101090980A (en)

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