CN101080238A - Compositions and methods for the preparation of human growth hormone glycosylation mutants - Google Patents
Compositions and methods for the preparation of human growth hormone glycosylation mutants Download PDFInfo
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Abstract
The present invention relates to a mutant of human growth hormone, the mutant include the newly introduced N-connected or O-connected glycosylation site, therefore the rescheduling polypeptides is provided with the glycosylation mode which is visibly different from the natural human growth hormone. Otherwise it opens the polynucleotide coding sequence of the mutant, the expression cassette including the coding sequence and the method for expressing the mutant cell and producing the mutant. Furthermore it opens the medicine composition including the mutant and the method for applying the mutant.
Description
Background of invention
The member that human growth hormone (hGH) and its agonist variant are recombinant protein families is as the U.S. patent No. 4,658,021 and the U.S. patent No. 5,633, described in 352.In the US. patent No. 4,342,832,4,601,980; U.S. the patent No. 4,898, and 830; U.S. the patent No. 5,424,199 and the U.S. patent No. 5,795,745 in describe their recombinant production and using method in detail.The human growth hormone participates in the many aspects of normal person's growth promoter regulation and control.By with its acceptor interaction, the pituitary hormone of this 22kDa is regulated many biological effects, for example the activation and the Insulin-Like of linear growth (body generation), suckling, macrophage and cause the diabetes effect.Chawla, Annu.Rev.Med., 34:519 (1983); Edwards etc., Science, 239:769 (1988); Isaksson etc., Annu.Rev.Physiol., 47:483 (1985); Thorner and Vance, J.Clin.Invest., 82:745 (1988); Hughes and Friesen, Annu.Rev.Physiol., 47:469 (1985).
In field of medicaments, give glycosylated and nonglycosylated peptide as everyone knows and can produce specific physiological reaction.Purification and reorganization hGH have been used for treating because disease and the disease that the hGH shortage causes, for example child's dwarfism.Limited an immunogenicity that principal element is most of peptides of the application of therapeutical peptide.In the patient, can make this peptide inefficacy and/or cause allergic formation among the patient the immunogenic response that gives polypeptide.The defective of other therapeutic glycopeptide comprises inferior optimum effectiveness and clearance rate fast.Prior art has been recognized the inherent problem of peptide therapeutics, and after deliberation eliminate the several different methods of this problem, for example,, synthetic polymer has been adhered to the peptide main chain for the soluble peptide therapy is provided.
Poly-(ethylene glycol) (" PEG ") is a kind of exemplary polymer that has been connected with polypeptide.Utilize PEG derived peptide therapy to show the immunogenicity that has reduced peptide.For example the U.S. patent No. 4,179, and 337 (Davis etc.) relate to the non-immunogenic polypeptide.Enzyme and the peptide hormone that links together with Polyethylene Glycol (PEG) or polypropylene glycol for example.Polymer between every mole of polypeptide has used 10 and 100 moles, and keep 15% physiologically active at least.In addition, the size increase causes the prolongation of loop cleaning time because described polypeptide is connected with PEG.Disclosed methods such as Davis are chemical Pegylation methods.
The chemical modification of peptide usually causes the active undesirable loss of peptide, is attributable to be used for the non-selective energy of the chemical action of modified polypeptide.For example, when modification group was water-soluble peptide such as PEG, the main pattern that PEG and its derivant are connected with peptide was the non-specific combination by the peptide ammino acid residue.To water-soluble polymer and interleukin-22 (Fisher etc., Br.J.Haematol., 82:654 (1992)), granulocyte colony-stimulating factor (Satake-Ishikawa etc., Cell Struct.Funct., 17:157 (1992)), tumor necrosis factor (Tsutsumi etc., Br.J.Cancer, 71:963 (1996)) and human growth hormone (Clark, Deng, J.Biol.Chem., 271:21969 (1996)) bonded research disclosed these proteinic chemical Pegylations and reduced the body inner recipient of this peptide in conjunction with activity.
In many chemical Pegylation methods, in fact Polyethylene Glycol be with randomly, nonspecific mode adds reactive residue on the peptide main chain.For the manufacture of therapeutic peptide, it obviously is desirable utilizing the derivatization strategy of product formation, that be easy to identify, homogenizing basically that causes specific marker.A kind of promising scheme for preparing the specific marker peptide is added the sugar moieties of modifying by utilizing enzymes such as glycosyl transferase to peptide.
Have regioselectivity and stereoselective advantage based on enzyme synthetic.And the synthetic unprotect substrate that uses of enzyme catalysis carries out.There is the enzyme of three kinds of primary categories to be used for the synthetic of carbohydrate, glycosyl transferase (for example, sialyltransferase, oligosaccharide transferring enzyme, N-acetylglucosaminyltrVnsferase) and glycosidase.Glycosidase further is categorized as exoglycosidase (for example, beta-Mannosidase, β-Pu Tangganmei) and endoglycosidase (for example, Endo-A, Endo-M).Each enzyme of these classifications all has been successfully used to synthetic carbohydrate.Summary as common sees also Crout etc., Curr.Opin.Chem.Biol.2:98-111 (1998).
Glycosyl transferase has been modified the oligosaccharide structure of glycopeptide, produces to have specific product good stereochemical and regional chemistry control.Prepare oligosaccharide and modify the carbohydrate structure that N-is connected with O-with glycosyl transferase, on the glycopeptide that particularly in mammalian cell, produces.For example, the terminal oligosaccharide of glycopeptide is fully sialylated and/or fucosylated, and more consistent sugared structure is provided, and has improved glycopeptide pharmacodynamics and various other biological and has learned characteristic.For example, adopt β-1, the illustration that the synthetic lactose amine of 4-galactosyltransferase is the application of glycosyl transferase aspect synthetic carbohydrate (for example referring to, Wong etc., J.Org.Chem.47:5416-5418 (1982)).And, many synthesis steps have utilized α-sialyltransferase that sialic acid (is for example transferred to the 3-OH of galactose or 6-OH from the single phosphoric acid of cytidine-5--N-n acetylneuraminic acid n, referring to Kevin etc., Chem.Eur.J.2:1359-1362 (1996)).Fucosyl transferase can be used for shifting fucose unit from guanosine-5 '-diphosphonic acid fucose to the specific hydroxyl of saccharide receptor.For example, Ichikawa has prepared sialic acid Lewis-X (Ichikawa etc., J.Am.Chem.Soc.114:9283-9298 (1992)) by the fucosyltransferase that relates to the clone with sialylated lactamide fucosylation.For the synthetic new development of the glycoconjugate that therapeutic uses is discussed, referring to Koeller etc., Nature Biotechnology 18:835-841 (2000), simultaneously referring to U.S. patent No. No.5,876,980; 6,030,815; 5,728,554; 5,922,577; And WO/9831826.
Glycosidase can also be used to preparing saccharide.The hydrolysis of the common catalysis glycosidic bond of glycosidase, however under appropriate condition, they can be used for forming this key.Being used for the synthetic most glycosidase of carbohydrate is exoglycosidase; The transfer of glycosyl occurs in the non-reduced end of substrate.Glycosidase obtains glycosyl donor in glycosyl-enzyme intermediate, produced hydrolyzate or produced new glucosides or oligosaccharide by receptor by the water interception.A kind of exemplary approach that utilizes exoglycosidase is the synthetic of all N-core trisaccharide of connecting glycopeptide, comprises the β-mannose glycosidic bond of difficulty, and it forms (Singh etc., Chem.Commun.993-994 (1996)) by the effect of beta-Mannosidase.
Form in the application of glycosidic bond at another exemplary glycosidase that utilizes, preparation mutant glycosidase wherein is converted to non-nucleophilic aminoacid with the normal nucleophilic aminoacid in the active site.This mutant enzyme is the hydrolysis sugar glycosidic bond not, but still can form them.Utilize alpha-glycosyl fluoride donor and glucosides acceptor molecule that the mutant glycosidase is used for preparing oligosaccharide (Withers etc., the U.S. patent No. 5,716,812).Although the mutant glycosidase has the purposes that forms free oligosaccharide, whether this kind of enzyme can append to glycosyl donor the proof that awaits on the glycosylated or nonglycosylated peptide, and these enzymes yet together do not use with non-activated glycosyl donor.
Although their application is lacked than exoglycosidase, endoglycosidase also is used to prepare carbohydrate.Method based on the utilization of endoglycosidase has following benefit, has promptly shifted oligosaccharide rather than monosaccharide.Used the endo-beta-N-acetyl glycosamine,, oligose fragment has been added to (Wang etc., Tetrahedron Lett.37:1975-1978) on the substrate as endo-F, endo-M; With Haneda etc., Carbohydr.Res.292:61-70 (1996)).
Except the application in the preparation carbohydrate, the above-mentioned enzyme that discusses also is used for the synthetic of glycopeptide.Delivered synthetic (Witte K. etc., the J.Am.Chem.Soc.119:2114-2118 (1997)) of the ribonuclease B of homogeneous sugar form.By handling the high mannose core that glycopeptide has divided ribonuclease B with endoglycosidase H.Division takes place between two core GlcNAc residues specifically.Pass through sequential use β-1 then, 4-galactosyltransferase, α-2,3-sialic acid conversion enzyme and α-1,3-fucosyltransferase V rebuilds tetrose sialic acid Lewis X with enzyme on the remaining GlcNAc anchor position point of homogeneous albumen.Each enzyme catalysis step is carried out with high yield.
The method of known combination chemistry and enzyme catalysis synthesized element in addition.For example, Yamamoto and its partner (Carbohydr.Res.305:415-422 (1998)) have reported and have used a kind of endoglycosidase to carry out glycopeptide be that the enzyme of glycosylated peptide T is synthetic.N-acetyl group glucamine peptide is synthetic by chemical method fully.Oligosaccharide with the human transferrin glycopeptide carries out enzymatically modifying to peptide subsequently.By saccharide partly being added to peptide chain with a kind of inscribe β-N-acetyl-glucosamine glycosidase.The glycosylated peptide that produces is highly stable, compares with N-acetyl group glucamine peptide T with peptide T, and proteolysis is had resistance.
After deliberation utilize glycosyl transferase with reporter group modified peptides structure.For example, (the U.S. patent No. 5 such as Brossmer, 405,753) application in measuring sialyltransferase active and cell surface, glycoprotein and ganglioside fluorescent labeling of the formation of fluorescently-labeled sialic cytosine riboside monophosphate (" CMP ") derivant and fluorescence glucosides is disclosed.Gross etc. (Analyt.Biochem.186:127 (1990)) have described a kind of similar test.People such as Bean (the U.S. patent No. 5,432,059) disclose a kind of with the glycosylated again glycosylation defect imbalance of insufficient glycosylated protein test.With defective protein with the glycosylation again of fluorescently-labeled CMP glucosides.Replace each fluorescence sialic acid derivative on the common acetylizad amine in site 9 or in sialic acid with the fluorescence part.The method of utilizing the fluorescence sialic acid derivative is to detect the test of the existence of the nonglycosylated or incorrect glycosylated glycoprotein of glycosyl transferase.This test in the sample of biological origin a small amount of enzyme or glycoprotein on implement.Enzymatic production in also not open or sialic preparation that hint utilization is modified or plant-scale glycosylation or the nonglycosylated peptide.
Enzymatic method also has been used for activating the glycosyl residue on the glycopeptide, carries out chemical modification subsequently.General using beta-Galactose oxidase activation glycosyl residue, it changes terminal galactose into corresponding aldehyde.This acetaldehyde links together with the modification group that contains amine subsequently.For example, people (Nature Biotech.19:142 (2001)) such as Casares is connected amycin with the chimeric oxidation galactose residue of recombinant MHCII-peptide.
Also modify glycosyl residue and be used for the load ketone group; for example; Mahal and its colleague (Science 276:1125 (1997)) have prepared N-acetyl propionyl (levulinoyl) mannosamine (" ManLev "), and it has ketone functional group in the site that natural substrate is occupied by acetyl group usually.Handle cell with ManLev, integrated ketone group at cell surface thus.In addition referring to people such as Saxon, Science 287:2007 (2000); People such as Hang, J.Am.Chem.Soc.123:1242 (2001); People such as Yarema, J.Biol.Chem.273:31168 (1998); And people such as Charter, Glycobiology 10:1049 (2000).
By several modes carbohydrate is connected on the glycopeptide, wherein the serine that is connected with mucin type O-of the agedoite that connects of N-and the mucin type O of threonine are maximally related for recombinant glycoprotein therapeutic agent.An initial determiner of proteinic glycosylation is a primary sequence, although other factors of apparent protein zone and conformation and so on also play a role.The glycosylation that N-connects occurs in consensus sequence NXS/T, and wherein X can be any aminoacid except that proline.
The method of above-mentioned discussion does not provide the approach of the modified peptides of a large amount of pharmacological activity that keeps their unaltered analog basically that is suitable on the acquisition industry.And this method does not allow to modify the site-specific combination of sugar to peptide or glycopeptide yet.This method does not provide the mode of preparation at the glycosylated or sugared modified peptides of puting together in non-natural site yet.
The present invention provides the glycosylation of these reorganization hGH mutant and/or the motility of sugared Pegylation to solve these needs by the N-that contains new importing is provided hGH mutant that connect or the glycosylation site that O-connects.And, the invention provides practical method on a kind of industry of modifying the mutant hGH peptide that N-or O-connect with modification group such as water-soluble polymer, therapeutic part, biomolecule or the like.Particularly importantly the mutant hGH that modifies in this method has the character of improvement, has improved its application as therapeutic or diagnostic medicament.
The invention summary
On the one hand, the invention provides a kind of isolating nucleic acid that contains encoding mutant body human growth hormone's polynucleotide sequence.This mutant human growth hormone comprises that non-existent N-connects or O-connection glycosylation site among the wild type human growth hormone.In some embodiments, the wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2.In some preferred versions, the mutant human growth hormone comprises SEQ ID NO:3,4,5,6,7,8 or 9 aminoacid sequence.
On the other hand, the invention provides and contain a kind of for example isolating expression of nucleic acids box of nucleic acid or cell that comprises encoding mutant body human growth hormone's polynucleotide sequence, this mutant human growth hormone comprises that non-existent N-connects or O-connection glycosylation site among the wild type human growth hormone.
On the other hand, the invention provides a kind of mutant human growth hormone, be included among the wild type human growth hormone that non-existent N-connects or O-connects glycosylated site.In some embodiments, the wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2.In some preferred versions, the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
On the other hand, the invention provides a kind of method of producing the mutant human growth hormone, this mutant human growth hormone is included in the glycosylation site that non-existent N-connects or O-connects among the wild type human growth hormone.This method comprises recombinant production mutant human growth hormone, and in new this mutant of glycosylation site glycosylation human growth hormone's step.In some embodiments, the wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2.In some preferred versions, the mutant human growth hormone is contained SEQ ID NO:3,4,5,6,7,8 or 9 aminoacid sequence.
On the other hand, the invention provides a kind of pharmaceutical composition with mutant human growth hormone of treatment effective dose, this mutant human growth hormone is included in the glycosylation site that non-existent N-connects or O-connects among the wild type human growth hormone.In some embodiments, the wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2.In some preferred versions, the mutant human growth hormone is contained SEQ ID NO:3,4,5,6,7,8 or 9 aminoacid sequence.
On the other hand, the invention provides a kind of method that the treatment human growth hormone lacks in the patient.This method comprise give the patient an amount of to treatment or improve the effective mutant human growth hormone of growth hormone deficiency.The mutant human growth hormone who is used for this method is included in non-existent N-connection of corresponding wild type human growth hormone or O-connection glycosylation site.In some embodiments, corresponding wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2.In some preferred versions, the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
Aspect each, the mutant human growth hormone is connected alternatively with one or more modification groups aforesaid, preferably is connected by means of sugar and produces the glycosyl linking group between glycosylation site and the modification group.A kind of exemplary modification group is poly-(ethylene glycol).
The accompanying drawing summary
Fig. 1 is the aminoacid sequence of GH-N (hGH in hypophysis source) and GH-V (hGH in Placenta Hominis source).Arrow shows the amino acid position of the glycosylation site of (GH-N) or naturally occurring (GH-V) N-connection that sudden change imports.
Fig. 2 is the crystal structure figure of glycosylated GH-N mutant hGH (Lys140 becomes Asn140) and its receptor polypeptides.
Fig. 3 is the sugared Pegylation scheme of the polysaccharide mutant that is connected of hGH N-that insect cell and mammalian cell are produced.
Fig. 4 has shown the sugared Pegylation of the polysaccharide mutant of the hGH O-connection that escherichia coli produce.
Fig. 5 has shown optional (alternate) mutant that imports the GH-N of glycosylation site.Arrow has shown the GH-N protein loop zone that can import glycosylation site.
Fig. 6 is the aminoacid sequence that six (6) the individual different O-that can import the hGH (GH-N) in hypophysis source connect glycosylation site.The wild-type amino acid sequence that has also shown GH-N is to compare.Arrow has shown, and the threonine residues that O-connects glycosylated GH-N polysaccharide mutant will take place.
Fig. 7 is that hGH O-connects GH-N mutant 134 (rtg) → ttt are connected 5 ' GH-N mutant with hGH O-aminoacid sequence, and wherein aminoacid-3 inserts amino terminal to-1 (ptt), causes producing 194 amino acid whose hGH polypeptide.
Fig. 8 is that hGH O-connects GH-N mutant 134 (rtg) → ttg are connected 5 ' GH-N mutant with hGH O-aminoacid sequence, and wherein aminoacid-3 inserts amino terminal to-1 (mvt), causes producing 194 amino acid whose hGH polypeptide.
Fig. 9 A has described the aminoacid sequence (SEQ ID NO:1) of ripe human growth hormone (GH-N).Fig. 9 B has described the aminoacid sequence (SEQ ID NO:2) of ripe human growth hormone (GH-V).Fig. 9 C has described the aminoacid sequence (SEQ ID NO:3) of human growth hormone's mutant 1.Fig. 9 D has described the aminoacid sequence (SEQ ID NO:4) of human growth hormone's mutant 2.Fig. 9 E has described the aminoacid sequence (SEQ ID NO:5) of human growth hormone's mutant 3.Fig. 9 F has described the aminoacid sequence (SEQ ID NO:6) of human growth hormone's mutant 4.Fig. 9 G has described the aminoacid sequence (SEQ ID NO:7) of human growth hormone's mutant 5.Fig. 9 H has described the aminoacid sequence (SEQ ID NO:8) of human growth hormone's mutant 6.Fig. 9 I has described the aminoacid sequence (SEQ ID NO:9) of human growth hormone's mutant 7.
Detailed Description Of The Invention
Definition
Term " nucleic acid " or " polynucleotides " refer to DNA (DNA) or ribonucleic (RNA) and the polymer thereof of strand or double chain form. Unless clearly limit, this term comprise contain known have with reference nucleic acid similarly be combined proterties and with the nucleic acid of the analog of the natural nucleotide of the mode metabolism that is similar to naturally occurring nucleotides. Unless otherwise stated, specific nucleotide sequence also implies and comprises its conservative variant (for example degenerate codon replacement), allele, straight homologues (Ortholog), SNPs and complementary series of modifying and the sequence that conclusivelys show. Specifically, the 3rd site of (or all) codons that can be by producing wherein one or more selections is with the replacement that mixes base and/or deoxyinosine residue and replace realizing degenerate codon (people such as Batzer, Nucleic Acid Res.19:5081 (1991); The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); And the people such as Rossolini, Mol.Cell.Probes 8:91-98 (1994)). The mRNA of term nucleic acid and gene, cDNA and gene code is used interchangeably.
Term " gene " expression relates to the dna fragmentation that produces polypeptide chain. It can be included in before the code area and zone afterwards (leading and afterbody) and the insertion sequence (introne) between absolute coding section (extron).
When being applied to nucleic acid or protein, term " separation " expression there is no nucleic acid or the protein at other cell parts of nature and its combination. It preferably is in homogeneous state, although can be the state of drying or the aqueous solution. The analysis such as normal operation such as polyacrylamide gel electrophoresis or high performance liquid chromatography chemical technology is determined purity and homogeneity. The protein of the kind that exists with advantage in goods is basic purifying. Particularly, the gene of separation separates with the opening code-reading frame of the albumen that is positioned at this gene flank and the non-genes of interest of encoding. Term " purifying " is illustrated in nucleic acid or the albumen that basically produces a band in the running gel. Refer specifically to this nucleic acid or albumen and have minimum 85% purity, more preferably at least 95% purity, most preferably at least 99% purity.
Term " amino acid " refers to natural existence and synthetic amino acid, and to be similar to natural amino acid analogue and the amino acid analog thing that exists amino acid whose mode to bring into play function. Naturally occurring amino acid is the amino acid by the genetic code coding, and the amino acid of modifying subsequently, for example, and hydroxyl (base) proline, Gla and O-phosphoserine. Amino acid analogue refers to have with naturally occurring amino acid the compound of identical basic chemical structure, namely, the α carbon of being combined with hydrogen, carboxyl, amino and R group, for example homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium etc. This analog has the R group (for example nor-leucine) of change or the peptide chain that changes, but has kept the basic chemical structure identical with naturally occurring amino acid. " amino acid analog thing " refers to have the compound that is different from the common chemical constitution of amino acid, but plays a role to be similar to naturally occurring amino acid whose mode.
There is in the prior art multiple known method non-natural amino acid derivativges or analog to be integrated into polypeptide chain in the locus specificity mode, for example referring to WO02/086075.
Three letter characters can being recommended by the usually known IUPAC-IUB biological chemical name method committee of amino acid or by a letter character representative herein. Equally, nucleotides can be represented by their alphanumeric codes of usually admitting.
" the conservative variant of modifying " is applied to aminoacid and nucleotide sequence simultaneously.For specific nucleotide sequence, " conservative modify variant " refer to encode nucleic acid of same or same basically aminoacid sequence maybe when this nucleic acid not during encoding amino acid sequence, is represented same basically sequence.Because the degeneracy of genetic code, identical nucleic acid coding given protein arbitrarily on many functions.For example, codon GCA, GCC, GCG and GCU coded amino acid alanine all.Thereby by the alanine site that codon is determined, this codon can be changed into above-mentioned any corresponding codon and not change encoded polypeptides at each.This nucleic acid variation is " silent variant ", is conservative change a kind of.Here each nucleic acid encoding sequence is also described the reticent variant of each possible nucleic acid.Those of skill in the art will recognize that each codon (except AUG, it is unique codon of methionine normally, and TGG, and it is the unique codon of tryptophan normally) in the nucleic acid all can change and produces molecule identical on the function.Correspondingly, the reticent variant of each of nucleic acid encoding all is implicitly included in the sequence of each description.
As for aminoacid sequence, those of skill in the art are a kind of " the conservative variants of modifying " with one displacement, disappearance or the interpolation of recognizing change in coded sequence, increase or deleting amino acid whose nucleic acid, peptide, polypeptide or the protein sequence of single amino acids or little percentage ratio, and wherein this change causes with similar aminoacid replacement one seed amino acid on the chemical property.The conservative substitution catalogue provides similar aminoacid on the function that is well known in the art.This conservative modification variant replenish and do not get rid of polymorphie variant of the present invention, plant between homologue and allele.
Following eight groups all contain the aminoacid of mutual conservative substitution:
1) alanine (A), glycine (G);
2) aspartic acid (D), glutamic acid (E);
3) agedoite (N), glutamine (Q);
4) arginine (R), lysine (K);
5) isoleucine (I), leucine (L), methionine (M), valine (V);
6) phenylalanine (F), tyrosine (Y), tryptophan (W);
7) serine (S), threonine (T); And
8) cysteine (C), methionine (M)
(for example referring to, Creighton, Proteins (1984)).
Herein aminoacid can recommend by common known IUPAC-IUB biological chemical name method committee they three alphabetic characters or by an alphabetic character representative.Equally, nucleotide can be represented by their letter code of admitting usually.
In this application, amino acid residue is according to its residue numbering with respect to the leftmost side in unaltered wild type peptide sequence, and the residue of this leftmost side is numbered 1.
" near proline residue " of herein adopting refers to proline residue at a distance of about 10 aminoacid with interior aminoacid, preferably with proline residue at a distance of about 9,8,7,6 or 5 with interior aminoacid, more preferably with proline residue at a distance of about 4,3,2 or 1 with interior residue.The aminoacid of " near proline residue " can be in the C or the N-terminal side of proline residue.
" polypeptide " of Cai Yonging, " peptide " and " protein " refer to the polymer of amino acid residue herein, are used interchangeably.These three terms are applicable to that all wherein one or more amino acid residues are amino acid polymers of the artificial chemical simulation thing of corresponding natural amino acid, and the amino acid polymer of naturally occurring amino acid polymer and non-natural existence.This term of Shi Yonging comprises the amino acid chain of any length herein, comprises full length protein, and wherein amino acid residue is connected by the covalency peptide bond.
Be used for importing term " sudden change " that additional N-or O-connect the situation of glycosylation site to the wild type human growth hormone and refer to carry out disappearance, the insertion of nucleotide arbitrarily or amino acid residue or replace at encoding wild type human growth hormone's polynucleotide sequence or wild type human growth hormone's aminoacid sequence respectively by chemistry, enzymatic or any other mode, consequent human growth hormone's aminoacid sequence contains at least one non-existent N-or O-connection glycosylation site in corresponding wild type human growth hormone.Under the situation of aminoacid replacement, conservative and non-conservative replacement all can be used to produce and contain the hGH mutant that new N-or O-are connected glycosylation site.
The site that imports the sudden change of new N-or O-connection glycosylation site can be positioned at any position of polypeptide.The aminoacid sequence of exemplary human growth hormone's mutant is described in SEQ ID NOs:3-9." mutant human growth hormone " of the present invention thereby contain at least one mutating acid residue.On the other hand, the wild type human growth hormone who has changed its coded sequence in this application and produced the mutant human growth hormone is called " corresponding wild type human growth hormone ", for example, SEQ ID NO:1 is corresponding wild type human growth hormone's the aminoacid sequence with mutant human growth hormone of aminoacid sequence SEQ ID NO:3-9.
The amount that produces therapeutic effects with a kind of material showed in the term of Shi Yonging " effective dose " herein.But this effect comprises prevention, rectification or the inhibition of any detection level of the symptom of disease/disease and relevant complication.Amount depends on the purpose of treatment accurately, and can by those skilled in the art adopt known technology determine (for example, referring to Lieberman, Pharmaceutical Dosage Forms (vols.1-3,1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); And Pickar, Dosage Calculations (1999)).
The carbohydrate that exists of the term of Shi Yonging " modify sugar " the natural or non-natural that refers in process of the present invention on the aminoacid of peptide or glycosyl residue, to add herein with Enzymology method.Modify sugar and select, includes but not limited to ribotide (single, double and triphosphoric acid), activation sugar (for example, glycosyl halide, glycosyl methanesulfonic acid) and the both also sugar of non-nucleotide of disactivation from many zymolytes.Employing " modification group " is covalently functionalized should " to modify sugar ".Available modification group includes but not limited to, water-soluble polymer, therapeutic part, diagnosis part, biomolecule or the like.Preferred and the non-natural existence of modification group, or a kind of unaltered carbohydrate.Select site, make it not stop " modifying sugar " enzymatic to add in the peptide with the modification group functionalization.
Term " water solublity " refers to have the part of certain detectable dissolubility in water.Water solublity detection and/or quantitative methods are well-known in the prior art.Exemplary water-soluble polymer comprises peptide, saccharide, poly-(ether), poly-(amine), poly-(carboxylic acid) or the like.Peptide can have the mixed sequence of being made up of single amino acids, for example poly-(lysine).A kind of exemplary polysaccharide is poly-(sialic acid), and a kind of exemplary poly-(ether) is poly-(ethylene glycol), for example, and m-PEG.Polymine is a kind of exemplary polyamine, and poly-(acrylic acid) is a kind of typical poly-(carboxylic acid).
The polymer backbone of water-soluble polymer can be poly-(ethylene glycol) (being PEG).Yet, know that very other related polymers also are suitable in the practice of the present invention, and the application in this respect of term PEG or poly-(ethylene glycol) is open surely and is not exclusive.Term PEG comprises any type of poly-(ethylene glycol), comprise alkoxyl PEG, bifunctional PEG, multi-arm PEG, fork PEG, branched PEG, tool side chain (pendent) PEG (promptly having one or more PEG or related polymers that hang on the functional groups of this polymer backbone), or wherein have the PEG of degradable key.
Polymer backbone can be collinear or branched.The branched polymer main chain is known usually in the prior art.Branched polymer generally has a center and props up the type core and prop up a plurality of linear polymer chains that the type core is connected with the center.PEG uses with the branch form usually, can be by adding the oxirane preparation to multiple polyhydric alcohol such as glycerol, tetramethylolmethane and Sorbitol.This center branch part can also be from several amino acid, for example lysine.Branch's poly-(ethylene glycol) can be expressed as usually R (PEG-OH) form of .sub.m., wherein R represents the core, for example glycerol or tetramethylolmethane, m represents the number of arm.For example the multi-arm PEG molecule of describing in the U.S. patent No. 5,932,462 also can be used as polymer backbone, and is here that the document is whole as the reference introducing.
Many other polymer also are suitable for the present invention.Have from 2 non-peptide class and water miscible polymer backbones and be specially adapted to the present invention to about 300 ends.The example of suitable polymer blend includes, but are not limited to other polyalkylene glycol, for example poly-(propylene glycol) (" PPG "), ethylene glycol and propylene glycol copolymers or the like, poly-(polyhydric alcohol of ethoxylation), poly-(enol), poly-(vinyl pyrrolidone), poly-(hydroxypropyl methyl acrylamide), poly-(alpha-hydroxy acid), poly-(vinyl alcohol), polyphosphazene, poly- azoles quinoline, poly-(N-acryloyl morpholine), for example in the U.S. patent No. 5,629, describe in 384, its integral body is incorporated herein by reference herein, and copolymer, terpolymer and its mixture.Although the molecular weight of each chain of polymer backbone can be different, generally at about 100Da to about 100, in the scope of 000Da, usually about 6,000Da is to about 80,000Da.
Being defined as for " area below the curve " or " AUC " that use in the situation with patient's peptide pharmaceutical products is herein describing as the medicine gross area below the body circulation concentration curve among the patients from the function of zero to infinitely-great time.
This be in to the situation of patient's peptide pharmaceutical products in the term " half-life " that uses or " t 1/2 " be defined as the plasma concentration of medicine in the patient and be reduced to the time that half needs.Have relevant with the peptide pharmaceutical products half-life more than one, it depends on multiple purge mechanism, redistributes and the well-known mechanism of other prior aries.Usually α and β half-life are defined as the α stage and redistribute relevantly, and the β stage is relevant with clearance rate.Yet the albumen medicine that is limited in the blood flow for major part can have at least two kinds to remove the half-life.To some glycosylated peptides, can mediate β stage clearance rate rapidly by macrophage or the receptor on the endotheliocyte of discerning terminal galactose, N-acetyl group galactosamine, N-acetyl-glucosamine, mannose or fucose.To special in the renal glomerular filtration of molecule with effective radius＜2nm (approximately 68kD) and/or the tissue or nonspecific picked-up and metabolism slower β stage clearance rate can take place via kidney.The sugar Pegylation can cover terminal sugar (for example, galactose or N-acetylgalactosamine), and therefore via discerning these sugared receptor blockings α stage clearance rate rapidly.May give and bigger effective radius in addition, thereby reduce volume and the tissue picked-up that distributes, thereby prolong the β stage in late period.Thereby sugared Pegylation changes along with size, glycosylation state and other parameters the influence accurately of α stage and β half-life in stage, as is known in the art.In Pharmaceutical Biotechnology, has further explanation (1997, DFA Crommelin and RD Sindelar, eds., HarwoodPublishers, Amsterdam, pp 101-120) to " half-life ".
The term of Shi Yonging " sugar is puted together " refers to that the aminoacid that will modify saccharide and polypeptide or the glycosyl residue of enzymatic mediation put together herein, for example, and mutant human growth hormone of the present invention.A subclass of " sugar is puted together " is " ethylene glycol-Pegylation ", and the modification group of wherein modifying sugar is poly-(ethylene glycol) and alkyl derivative (for example m-PEG) or its reactive derivant (for example H2N-PEG, HOOC-PEG).
Term " on a large scale " and " commercial scale " are used interchangeably, and refer to finish produce at least approximately 250mg single reaction time, preferably about at least 500mg, the reaction time of the glycoconjugate of more preferably about at least 1 gram.
The term of Shi Yonging " glycosyl linking group " glycosyl residue that referred to modification group (for example peg moiety, therapeutic part, biomolecule) covalently bound herein; This glycosyl linking group is connected to modification group the remainder of conjugate.In the method for the invention, " glycosyl linking group " covalently is connected with glycosylated or not glycosylated peptide, thereby this reagent is connected on the aminoacid and/or glycosyl residue of peptide." glycosyl linking group " is by will " modify sugar " being connected and deriving from " modification is sugared " usually with the aminoacid of enzyme and peptide and/or glycosyl residue.The glycosyl linking group can be the saccharide derived structure of a degraded (for example oxidation → Schiff's base formation → reduction) during modification group-modification sugar bowl forms, or this glycosyl linking group can be kept perfectly." the glycosyl linking group that is kept perfectly " refers to derive from the linking group of glycosyl part, wherein connects the not degraded of sugar monomer of the remainder of modification group and conjugate, for example oxidation, for example, by the sodium metaperiodate oxidation." complete glycosyl linking group " of the present invention can derive from the oligosaccharide that exists natively, promptly adds glycosyl unit or removes one or more glycosyl units from parental generation sugar structure.
The term of Shi Yonging " targeting moiety " refers to optionally be positioned the kind in specific tissue of body or zone herein.The location is by mediations such as the molecular size of specific recognition, targeting agent or the conjugate of molecule determinant, ionic interaction, hydrophobic interactions.Directed to give other mechanism with specific tissue or zone be that those skilled in the art are known with reagent.Exemplary targeting moiety comprises antibody, antibody fragment, transferrin, HS-glycoprotein, thrombin, serum albumin, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO etc.
Any reagent that can be used for treating of " therapeutic part " expression that herein uses includes but not limited to antibiotic, antibiotic medicine, antineoplastic agent, cytotoxin and radioreagent." therapeutic part " comprises the prodrug of bioactive agents, wherein by surpassing the construct that a therapeutic partly is incorporated into carrier, for example, multivalence reagent.
The therapeutic part also comprises protein and comprises proteinic construct.Exemplary protein includes, but are not limited to erythropoietin (EPO), granulocyte colony-stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), interferon (interferon-' alpha ' for example, β, γ), interleukin (for example interleukin II), serum albumin (proconvertin for example, VIIa, VIII, IX and X), human chorionic gonadotropin (HCG), follicle stimulating hormone (FSH) and luteotropic hormone (LH) and antibody fusion protein (for example Tumor Necrosis Factor Receptors (TNFR)/Fc domain fusion rotein)).
" antineoplastic agent " that uses represented any anticancer reagent that can be used for herein, includes but not limited to cytotoxin and for example reagent of antimetabolite, alkylating agent, anthracycline, antibiotic, antimitotic agent, procarbazine, hydroxyurea, asparaginase, corticosteroid, interferon and radioreagent and so on.Being also included within the scope of term " antineoplastic agent " is the peptide conjugate with anti-tumor activity, for example TNF-α.Conjugate includes, but are not limited to the conjugate that forms between therapeutic protein of the present invention and glycoprotein, a kind of typical conjugate is to form between PSGL-1 and the TNF-α.
" cytotoxin or the cytotoxin reagent " of Shi Yonging refers to the deleterious any reagent of pair cell herein.Example comprises paclitaxel, cytochalasin B, Gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide, tenoposide, vincristin, vincaleucoblastine, colchicine, amycin, daunorubicin, dihydroxy anthracinedione, mitoxantrone, plicamycin, actinomycin D, 1-boldenone, glucocorticoid, procaine, tetracaine, lignocaine and puromycin and its analog or congener.Other toxin comprises that as ricin, CC-1065 and analog, amycin other toxin comprises diphtheria toxin, diphtherotoxin and snake venom (for example cobra venom).
Herein " radioreagent " of Shi Yonging be included in tumor make a definite diagnosis or destroy in effective any radiosiotope.Example includes, but are not limited to indium-111, cobalt-60.In addition, naturally occurring radioelement, for example the uranium of the radioisotopic mixture of general proxy, radium and thorium are the examples of the radioreagent that suits.General and the organic chelated part chelating of metal ion.
Many useful chelation groups known in the state of the art, crown ether, cryptand or the like, they (for example can incorporate chemical compound of the present invention into, EDTA, DTPA, DOTA, NTA, HDTA or the like and phosphonate analogs thereof, for example DTPP, EDTP, HDTP, NTP or the like), for example referring to, Pitt etc., " The Design of Chelating Agents for the Treatmentof Iron Overload ", In, INORGANIC CHEMISTRY IN BIOLOGY ANDMEDICINE; Martell, Ed.; American Chemical Society, Washington, D.C., 1980, pp.279-312; Lindoy, THE CHEMISTRY OF MACROCYCLICLIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989; Duga s, BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, and the list of references that wherein contains.
In addition, those skilled in the art are known has number of ways chelating agen, crown ether and cyclodextrin can be connected with other molecule.For example, referring to, Meares etc., " Properties of In VivoChelate-Tagged Proteins and Polypeptides ", In, MODIFICATIONOF PROTEINS:FOOD, NUTRITIONAL, AND PHARMACOLOGICAL ASPECTS; Feeney etc., Eds., American Chemical Society, Washington, D.C., 1982, pp.370-387; Kasina etc., Bioconjugate Chem., 9:108-117 (1998); Song etc., Bioconjugate Chem., 8:249-255 (1997).
" the pharmaceutically acceptable carrier " of Shi Yonging comprises the activity that keeps conjugate when making up with conjugate herein, and to the responseless any material of patient's immune system.Example includes, but are not limited to any standard drug carrier, for example phosphate buffered salt solution, water, as Emulsion and various types of wetting agent of oil/aqueous emulsion.Other carrier also can comprise sterile solution, tablet (comprising sugar coated tablet) and capsule.Common this carrier contains excipient, as clay, gelatin, stearic acid or its salt, calcium stearate or magnesium, Talcum, plant fat or oil, natural gum, dihydroxylic alcohols or other known excipient of starch, milk, sugar, certain type.This carrier also can comprise fragrance and color additives or other composition.The compositions that contains this carrier is prepared with well-known traditional methods.
" administration " expression of herein using is oral, suck, as suppository administration, local contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or the implantation of delayed release device is for example adopted little osmotic pumps to the patient.Administration can be passed through approach arbitrarily, comprises non-intestinal and through mucous membrane (for example, mouth, nose, vagina, rectum or transdermal).The parenteral method for example comprises in intravenous, intramuscular, intra-arterial, Intradermal, subcutaneous, intraperitoneal, the ventricle and intracranial.And for example apoptosis-induced when injection is the treatment tumor, administration can directly be given with tumor and/or be given and tumor tissue on every side.Other administering mode includes, but are not limited to utilize Liposomal formulation, intravenous infusion, transdermal patch etc.
Term " isolating " refers to fully or is substantially free of the material of some composition, and wherein said composition is used for producing this material.To peptide conjugate of the present invention, this term " isolating " refers to fully or is substantially free of the material of some component, and described component is accompanied by the material of the mixture that is used for preparing peptide conjugate usually." separation " and " purification " can exchange use.Usually, isolating peptide conjugate of the present invention has the preferred purity level of the certain limit of being expressed as.Peptide conjugate purity range following is limited to about 60%, about 70% or about 80%, and the upper limit of purity range is approximately 70%, about 80%, about 90% or be higher than about 90%.
When peptide conjugate was higher than about 90% purity, their purity also preferably was expressed as certain scope.The following of this purity range is limited to about 90%, about 92%, about 94%, about 96% or about 98%.Be limited to about 92%, about 94%, about 96%, about 98% or about 100% on this purity range.
Purity is determined (for example, silver dyes band intensity, polyacrylamide gel electrophoresis, HPLC or the similar mode on the gel) by any analytical method known in the art.
" each member basically of this colony " of Shi Yonging described the feature of the colony of peptide conjugate of the present invention herein, and the modification sugar on the peptide of being added to that wherein will select ratio is added to a plurality of identical acceptor site on the peptide chain." each member basically of this colony " refer to and the homogeneity of modifying site on the peptide that sugar puts together, and refers to conjugate of the present invention minimum about 80%, preferred about at least 90% and more preferably about at least 95% homogeneity.
" homogeneity " refers to modify the structural integrity of the acceptor portion colony that sugar puts together.Therefore, in peptide conjugate of the present invention, each modifies other modification acceptor site that sugar moieties connected of acceptor site and each that sugar moieties connected when identical, and then this peptide conjugate is about 100% homogeneous.Homogeneity is typically expressed as a scope, and peptide conjugate homogeneity scope following is limited to about 60%, about 70% or about 80%, and the upper limit of purity range is approximately 70%, about 80%, about 90% or be higher than about 90%.
When peptide conjugate is greater than or equal to about 90% all for the moment, their homogeneity also preferably is expressed as certain scope.The following of this homogeneity scope is limited to about 90%, about 92%, about 94%, about 96% or about 98%.Be limited to about 92%, about 94%, about 96%, about 98% or about 100% on the purity range.The purity of peptide conjugate is determined by one or more methods known to those skilled in the art that usually for example, liquid chromatography-mass spectrography is learned (LC-MS), substance assistant laser desorpted time-of-flight mass spectrometry (MALDITOF), capillary electrophoresis or the like.
When relating to glycopeptide kind time-like, " unified substantially sugar form " or " unified substantially glycosylation pattern " refers to the percentage ratio by the glycosylated acceptor portion of purpose glycosyl transferase (for example fucosyltransferase).For example, for α 1,2 fucosyltransferases, if whole basically (as hereinafter defined) Gal β 1 in peptide conjugate of the present invention, 4-GlcNAc-R and its sialylated analog are fucosylated, then have unified basically fucosylation pattern.It will be understood by those skilled in the art that parent material can contain glycosylated acceptor portion (for example fucosylated Gal β 1,4-GlcNAc-R part).Therefore, the glycosylation percentage ratio of calculating comprises by the glycosylated acceptor portion of method of the present invention, and in parent material glycosylated acceptor portion.
Term " basically " ordinary representation in " unified substantially " of above-mentioned definition about at least 40%, about at least 70%, about at least 80% or more preferably about at least 90%, more preferably the acceptor portion of about at least 95% specific glycosyl transferase is glycosylated.
Apparent other object of the present invention, aspect and advantage from following detailed description.Abbreviation
PEG, poly-(ethylene glycol); M-PEG, methoxyl group-poly-(ethylene glycol); PPG, poly-(propylene glycol); M-PPG, methoxyl group gathers (propylene glycol); Fuc, fucosido; Gal, galactosyl; GalNAc, N-acetyl galactosyl amino; Glc, glucityl; GlcNAc, N-acetyl group glucamine; Man, mannose group; ManAc, the mannose aminoacetic acid; Sia, sialic acid; And NeuAc, the N-acetylneuraminic amine.
Brief introduction
In order to improve the recombinant human somatropin's who is used for the treatment of purpose effect, the invention provides human growth hormone's genetically engineered mutant body, contain the glycosylation site that non-existent N-connects or O-connects among the naturally occurring human growth hormone.Though these hGH mutants have kept the biological activity of wild type hormone basically, the new glycosylation site that imports makes the hGH mutant of reorganization generation with the various modes glycosylation.And non-natural glycosylation site provides the site of puting together of peptide and modification group, for example, puts together by sugar.A kind of exemplary modification group is a water-soluble polymer, and for example poly-(ethylene glycol) is as methoxyl group poly-(ethylene glycol).Modify the hGH mutant and can improve stability and the retention time of recombinant hGH in patient's circulation, reduce their antigenicity, and improve the ability of the particular organization that their targeting need treat.
Mutant
The invention provides the mutant of hGH, it is included in undiscovered one or more O-or N-connection glycosylation site in the wild type peptide.This mutant is usually can glycosylation in wild type peptide, or the glycosylated substrate of enzymatic on may seldom glycosylated one or more sites, therefore, this mutant site of having changed glycosyl residue or glycosyl linking group is with the peptide of the desirable character that obtains to have selection.Except the site and number of glycosyl residue or glycosyl linking group, other character that can utilize mutant of the present invention and method to change comprise pharmacokinetics, pharmacodynamics, the resistance to proteolysis, immunogenicity, the identification by the reticuloendothelium system, tissue distribution or the like.
Therefore, the invention provides a kind of isolating nucleic acid that contains encoding mutant body human growth hormone's polynucleotide sequence on the one hand.This mutant human growth hormone is included in non-existent N-connection or O-connection glycosylation site among the corresponding wild type human growth hormone.In some embodiments, corresponding wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2, in some preferred versions, the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
On the other hand, the invention provides and contain a kind of expression cassette or cell that comprises the nucleic acid (for example isolating nucleic acid) of encoding mutant body human growth hormone's polynucleotide sequence, this mutant human growth hormone is included in non-existent N-connection or O-connection glycosylation site among the corresponding wild type human growth hormone.
On the other hand, the invention provides a kind of mutant human growth hormone, it comprises the glycosylation site that non-existent N-connects or O-connects among the wild type human growth hormone.In some embodiments, corresponding wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2, in some preferred versions, the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
On the other hand, the invention provides a kind of method of producing the mutant human growth hormone, this mutant comprises among the corresponding wild type human growth hormone that non-existent N-connects or O-connects glycosylated site.This method comprises recombinant production mutant human growth hormone, and in new this mutant of glycosylation site glycosylation human growth hormone's step.In some embodiments, corresponding wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2.In some preferred versions, the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
The acquisition of hGH coded sequence
Conventional recombinant technique
The present invention is based on genetic recombination and learn the routine techniques in field.Basic document discloses the conventional method that adopts among the present invention, comprises Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed.2001); Kriegler, Gene Transfer andExpression:A Laboratory Manual (1990); And Ausubel etc., eds., Current Protocols in Molecular Biology (1994).
For nucleic acid, to (kb) or base pair (bp) expression, these are all from agarose or acrylamide gel electrophoresis, the nucleic acid of order-checking or the estimation of disclosed DNA sequence with kilobase for size.For protein, size represents that with kilodalton (kDa) or total number of atnino acid the protein size is all estimated from the albumen of gel electrophoresis, order-checking, the aminoacid sequence or the disclosed protein sequence of acquisition.
The oligonucleotide that can not buy from the market is synthetic chemically, for example, and according to Beaucage ﹠amp; Caruthers, the solid phase phosphoramidite three ester methods of at first describing among the Tetrahedron Lett.22:1859-1862 (1981), the automatic synthesizer that use Van Devanter etc. describes in Nucleic Acids Res.12:6159-6168 (1984).The purification of oligonucleotide can use arbitrarily the prior art known strategy to carry out, for example Pearson ﹠amp; Natural acrylamide gel electrophoresis or anion exchange HPLC that Reanier describes in J.Chrom.255:137-149 (1983).
The sequence, encoding mutant body human growth hormone's polynucleotide and synthetic oligonucleotide of clone's wild type human growth hormone gene can verify behind the clone, for example uses the chain termination method of the double-stranded template order-checking among the Gene 16:21-26 (1981) such as Wallace.
The clone and the sub-clone of wild type hGH coded sequence
Some encoding wild type human growth hormones' polynucleotide sequence, for example GenBank accession number NM 000515, NM 002059, NM 022556, NM 022557, NM 022558, NM 022559, NM 022560, NM 022561 and NM 022562 have determined also can obtain from commercial provider.
Rapid progress to the research of people's gene group has obtained possible cloning process, wherein can in the human DNA sequence data base, search for known nucleotide sequence have the genetic fragment arbitrarily of certain sequence homology percentage ratio, for example one the coding known human growth hormone gene.So the DNA sequence arbitrarily of identification can be subsequently by chemosynthesis and/or for example overlap extension method acquisition of polymerase chain reaction (PCR) technology.For short sequence, fully all de novo synthesis; Yet using synthetic probe further to separate complete encoding sequence from people cDNA or genomic library may be essential to obtaining bigger gene.
In addition, coding human growth hormone's nucleotide sequence can operating specification clone technology for example polymerase chain reaction (PCR) from people cDNA or genome dna library, separate, wherein often can come own coding human growth hormone's known nucleic acid based on the primer of homology.For this purpose the technology of normal use in standardized books, describe to some extent, for example, Sambrook and Russell, the same.
The cDNA library that is applicable to acquisition wild type human growth hormone coded sequence can have been bought maybe and can make up on the market.Separating mRNA, by reverse transcription produce cDNA, connect that cDNA enters recombinant vector, is transfected into that recombinant host is bred, examination and clone's conventional method is well-known (for example referring to Gubler and Hoffman, Gene, 25:263-269 (1983); Ausubel etc., the same).After the fragment of the nucleotide sequence that obtains amplification by PCR, this fragment can further be separated encoding wild type human growth hormone's total length polynucleotide sequence from the cDNA library as probe.The general remark of suitable step can be at Sambrook and Russell, finds in the same.
Can carry out the full length sequence that similar step obtains the encoding wild type human growth hormone from people's gene group library, for example the sequence of any one above-mentioned GenBank accession number.People's gene group library can be bought acquisition from the market, or can make up according to the known method of multiple prior art.Usually, for making up genomic library, at first from a kind of tissue that may find the human growth hormone, extract DNA, then this DNA is mechanically cut off or produces with enzymic digestion the fragment of about 12-20kb length, by gradient centrifugation isolated fragment and insert the phage carrier from the polynucleotide passage of undesired size, these carriers and phage are in external packing subsequently.By Benton and Davis, the plaque hybridization that Science, 196:180-182 (1977) describe is analyzed recombinant phage, and colony hybridization is according to Grunstein etc., the carrying out of describing among the Proc.Natl.Acad.Sci.USA, 72:3961-3965 (1975).
Based on sequence homology, it is right as primer to design degenerate oligonucleotide, and can carry out under optimum conditions PCR (for example referring to people such as White, PCR Protocols:CurrentMethods and Applications, 1993; Griffin and Griffin, PCRTechnology, CRC Press Inc.1994) come from cDNA or genomic library amplification of nucleotide sequence fragment.The fragment of using amplification obtains encoding wild type human growth hormone's total length nucleic acid as probe.
After obtaining encoding wild type human growth hormone's nucleotide sequence, the coded sequence sub-clone can be gone into carrier, for example, therefore a kind of expression vector can produce reorganization wild type human growth hormone from the construct that obtains.Can further change wild type human growth hormone's coded sequence subsequently, for example, carry out nucleotide and replace to change the characteristic of molecule.
In the hGH sequence, import sudden change
Can determine wild type human growth hormone's aminoacid sequence from a kind of coded polynucleotide sequence, for example, SEQ ID NO:1 or SEQ ID NO:2.Subsequently, can modify these aminoacid sequences to change proteinic glycosylation pattern by importing other glycosylation site on the diverse location in aminoacid sequence.
The protein glycosylation site of well-known several types in the prior art, for example, in eukaryote, N-connects glycosylation and occurs in consensus sequence Asn-X
AaOn the agedoite of-Ser/Thr, X wherein
AaBe aminoacid arbitrarily (Kornfeld etc., the Ann Rev Biochem 54:631-664 (1985) except that proline; Kukuruzinska etc., Proc.Natl.Acad.Sci.USA 84:2145-2149 (1987); Herscovics etc., FASEB J7:540-550 (1993); And Orlean, Saccharomyces Vol.3 (1996)).O-connect glycosylation occur on serine or the threonine residues (Tanner etc.,, Biochim.Biophys.Acta.906:81-91 (1987); And Hounsell etc., Glycoconj.J.13:19-26 (1996)).Other glycosylation pattern is connected with proteinic carboxyl terminal carboxyl by glycosyl-phosphatidyl inositol and forms (Takeda etc., Trends Biochem.Sci.20:367-371 (1995); And Udenfriend etc., Ann.Rev.Biochem.64:593-591 (1995).Based on these knowledge, therefore can introduce suitable sudden change to form new glycosylation site to wild type human growth hormone sequence.
New N-connects or O-connects glycosylation site although the direct change of the amino acid residue in human growth hormone's peptide sequence can be suitable for importing, yet more situation is to import new glycosylation site usually by means of the sudden change of coding human growth hormone's polynucleotide sequence.This can realize wherein having some to discuss hereinafter by using any known method of mutagenesis.Exemplary human growth hormone's change comprises and illustrating among SEQ ID NO:3 or the SEQ ID NO:4.
The method of multiple generation sudden change has been described in the prior art.For example, referring to, Zhang etc., Proc.Natl.Acad.Sci.USA, 94:4504-4509 (1997); And Stemmer, Nature, 370:389-391 (1994).This method can respectively or be united the variant that is used to produce one group of nucleic acid, and encoded polypeptides variant thus.The test kit of mutation, library construction and other multiformity production methods all can be bought from the market and obtain.
For instance, produce multifarious mutation method and comprise direct mutagenesis (Botstein and Shortle, Science, 229:1193-1201 (1985)), use contains the mutation (Kunkel of the template of uracil, Proc.Natl.Acad.Sci.USA, 82:488-492 (1985)), mutation (Zoller and Smith that oligonucleotide instructs, Nucl.Acids Res., 10:6487-6500 (1982)), DNA mutation (Taylor etc., the Nucl.Acids Res. of thiophosphate modification, 13:8749-8764 and 8765-8787 (1985)), and the mutation (Kramer etc., Nucl.Acids Res., 12:9441-9456 (1984)) of using the breach double-stranded DNA.
Other suitable method that produces sudden change comprises a mispairing reparation (Kramer etc., Cell, 38:879-887 (1984)), use and repair the mutation (Carter etc. that lack limit host strain, Nucl.Acids Res., 13:4431-4443 (1985)), deletion mutagenesis (Eghtedarzadeh and Henikoff, Nucl.Acids Res., 14:5115 (1986)), restriction is selected and restriction purification (Wells etc., Phil.Trans.R.Soc.Lond.A, 317:415-423 (1986)), by the synthetic mutation (Nambiar etc. of total gene, Science, 223:1299-1301 (1984)), the double-stranded reparation (Mandecki that interrupts, Proc.Natl.Acad.Sci.USA, 83:7177-7181 (1986)), polynucleotide chain termination method mutation (the U.S. patent No. 5,965,408), and error-prone PCR (Leung etc., Biotechniques, 1:11-15 (1989)).
Change nucleic acid to be suitable for the preference codon utilization in the host organisms
The polynucleotide sequence that can change encoding mutant body human growth hormone in addition is to meet the preference codon utilization of specific host.For example, the utilization of bacterial cell strain preference codon can be used for drawing coding mutant human growth hormone's of the present invention polynucleotide, and comprises the codon of this bacterial strain preference.The frequency that the preference codon that host cell demonstrates utilizes can be calculated (for example, the network address from Japanese Kazusa DNA institute can obtain calculation services) by the average frequency that is utilized by the preference codon in a large amount of gene of host cell expression.These are analyzed and preferably are limited to the gene that the host cell height is expressed.For example the U.S. patent No. 5,824,864 frequencies that provide the codon of the cance high-expression gene that dicotyledon and monocotyledon demonstrate to use.
After modification is finished, determine mutant human growth hormone's coded sequence by checking order, sub-clone is gone into a kind of suitable expression vector to carry out recombinant production with the same method of wild type human growth hormone then.
The expression of mutant hGH and purification
After order-checking confirmed, the routine techniques that can use genetic recombination to learn the field relied on the polynucleotide sequence of coded polypeptide disclosed herein to produce mutant human growth hormone of the present invention.
Expression system
For the high level expression of the mutant human growth hormone's of the present invention that obtains to encode nucleic acid, usually encoding mutant body human growth hormone's polynucleotide sub-clone is gone into a kind of contain instruct the strong promoter of transcribing, transcribe/expression vector of the ribosome binding site of translation termination and transcription initiation in.The promoter of well-known suitable antibacterial in the prior art, for example Sambrook and Russell see above, and Ausubel etc., see above and wait all description to some extent.The bacterial expression system of expressing wild type or mutant human growth hormone all can obtain at escherichia coli, bacillus cereus, Salmonella and Caulobacter etc.The test kit that is used for this expression system can be bought on market and obtain.The eukaryotic expression system of mammalian cell, yeast and insect cell is well-known in the prior art, also can buy acquisition from the market.In a specific embodiment, the eukaryote expression vector is adenovirus vector, adeno-associated virus vector or retroviral vector.
Be used to instruct the promoter of heterologous expression of nucleic acid to depend on specific application.The distance of promoter and heterologous transcriptional start site alternatively with in its natural surroundings at a distance of the distance of transcriptional start site much at one.Yet as be known in the art, can allow some changes of this distance and do not lose promoter function.
Except promoter, this expression vector generally includes transcript unit or expression cassette, contains to express the needed whole add ons of mutant human growth hormone in host cell.Therefore typical expression cassette contains the promoter that is connected with encoding mutant body human growth hormone's nucleotide sequence operability ground and transcript signal, ribosome binding site and the translation termination site of polyadenylation needs effectively.Coding human growth hormone's nucleotide sequence is connected with the signal peptide sequence of cleavable usually, to promote transformant secretion human growth hormone.The sort signal peptide comprises from tissue plasminogen activator, insulin and the neure growth factor, and the signal peptide of tobacco budworm (Heliothis virescens) JH esterase.Other element of this box can comprise enhancer, and if genomic DNA as structural gene, then can also comprise intron with functional donor splicing site and acceptor site.
Except promoter sequence, also contain the tanscription termination zone in the downstream of expression cassette structural gene, so that effective termination to be provided.Stop the zone can with promoter sequence available from identical gene, maybe can from different genes, obtain.
Be used for hereditary information is transported to the strictness especially of specific expression vector of cell, can use any conventional carrier that is used at eucaryon or procaryotic cell expression.The standard bacterial expression vector for example comprises the plasmid of plasmid based on pBR322, pSKF, pET23D and so on and the amalgamation and expression system of GST and LacZ for example.Can also add the antigenic determinant label to recombinant protein matter, c-myc for example is to provide isolating facilitated method.
The expression vector that contains from the controlling element of eucaryon virus is generally used for Eukaryotic expression vector, for example SV40 carrier, papillomatosis poisonous carrier and the carrier that derives from Epstein-Barr virus.Other exemplary eukaryotic vector comprises pMSG, pAV009/A, pMT010/A, pMAMneo-5, baculovirus pDSVE and other carrier arbitrarily that allows protein expression under SV40 early promoter, SV40 late promoter, metallothionein promoter, murine marrmary tumor virus promoter, rous sarcoma virus promoter, polyhedrin promoter or other promoter that is presented at effective expression in the eukaryotic cell instruct.
The label of the gene amplification of providing is provided some expression systems, as thymidine kinase, hygromycin B phosphotransferase and dihydrofolate reductase.In addition, the high yield expressing system that does not relate to gene amplification is also suitable, and the baculovirus vector in the insect cell for example has the polynucleotide sequence of the encoding mutant body human growth hormone under polyhedrin promoter or other strong bacilliform virus promoter instruct.
The element that is generally comprised within the expression vector is also included within the replicon of functionating in the escherichia coli, the gene of antibiotic resistance so that the antibacterial that contains recombiant plasmid is selected of encoding, and at unique restriction site of the nonessential region of plasmid, to allow the insertion of eukaryote sequence.The specific antibiotics resistance gene of selecting is not strict, and any many resistant genes well known in the prior art all suit.If necessary, select the prokaryote sequence, make them not interfere duplicating of DNA in the eukaryotic cell.Be similar to the antibiotic resistance selectable marker, also can be with the means of the transformed host cells that elects based on the metabolism selectable marker of known metabolic pathway.
When needing the periplasmic expression of recombinant protein (hGH mutant for example of the present invention), expression vector contains the sequence of the secretion signal of encoding in addition, for example escherichia coli OppA (the pericentral siphon oligopeptide is conjugated protein) secretion signal or its modified forms, its directly with 5 of the coded sequence of expressed protein ' be connected.This signal sequence instructs the recombinant protein that produces in the Cytoplasm to enter periplasmic space by cell membrane.This expression vector can comprise the coded sequence of signal peptidase 1 in addition, and it can enzymatic heading signal sequence when recombinant protein enters periplasmic space.Being described in more detail of the pericentral siphon production of recombinant protein can be at Gray etc., and Gene 39:247-254 (1985), the U.S. patent No. 6,160,089 and 6,436 find in 674.
As discussed above, those skilled in the art will realize that and can carry out various conservative replacements, and still keep human growth hormone's biological activity wild type or mutant human growth hormone or its coded sequence.And, also can utilize situation to carry out the modification of polynucleotide encoding sequence according to the preference codon in the specific expressive host, and not change the aminoacid sequence of generation.
Transfection method
Mass mutation body human growth hormone's antibacterial, mammal, yeast, insecticide or plant cell expressed in the production of employing standard transfection method, use then the standard technique purification (for example referring to, Colley etc., J.Biol.Chem.264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, vol.182 (Deutscher, ed., 1990)).According to standard technique carry out eukaryote and prokaryotic cell conversion (for example referring to, Morrison, J., Bact.132:349-351 (1977); Clark-Curtiss ﹠amp; Curtiss, Methods in Enzymology 101:347-362 (Wu etc., eds, 1983).
Can use any well-known method that in host cell, imports the foreign nucleus nucleotide sequence.This comprises and utilizes calcium phosphate transfection, 1,5-dimethyl-1,5-phenodiazine 11 methylene gather Methobromide, protoplast fusion, electroporation, liposome, microinjection, blood plasma carrier, viral vector and in host cell, import cloned genes group DNA, cDNA, synthetic DNA or other external hereditary material any other well-known method (for example, referring to, Sambrook and Russell, as mentioned above).Unique needs a bit be: specific genetic engineering method can successfully import at least one gene to the host cell that can express the mutant human growth hormone.
In host cell, detect the expression of mutant hGH
After expression vector is imported into proper host cell, under the condition that helps mutant human growth hormone expression, cultivate cells transfected, screen recombinant polypeptide expression in the cell then, use subsequently standard technique from culture medium, reclaim the recombinant polypeptide (for example referring to, Scopes, Protein Purification:Principles and Practice (1982); U.S. the patent No. 4,673, and 641; Ausubel etc., as mentioned above; And Sambrook and Russell are as mentioned above).
Some conventional methods that the well-known screening-gene of those skilled in the art is expressed.At first, can detect gene expression, use many special DNA and the RNA assay method (for example Sambrook and Russell see above) that utilizes nucleic acid hybridization technique usually in nucleic acid level.Certain methods relates to electrophoretic separation (for example detecting the Southern trace of DNA and the Northern trace of detection RNA), but the detection of DNA or RNA also can not carried out electrophoresis (for example passing through Dot blot).The existence of encoding mutant body human growth hormone's nucleic acid can also utilize sequence-specific primer to detect by PCR or RT-PCR in the cells transfected.
The second, can be in the gene expression of polypeptide horizontal detection.Those skilled in the art adopt the panimmunity credit to analyse the level of measuring gene outcome usually, particularly utilize polyclone or the monoclonal antibody that acts on specifically with mutant human growth hormone of the present invention, the polypeptide that for example has SEQ ID NO:3, an aminoacid sequence of 4 or 5 (for example, Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256:495-497 (1975)).This Technology Need is by selecting the antibody with high specificity of specificity at mutant human growth hormone or its antigen part.Well set up production polyclone and monoclonal antibody method, their explanation can be found in the literature, for example referring to Harlow and Lane, sees above; Kohler and Milstein, Eur.J.Immunol., 6:511-519 (1976).Part subsequently will provide antibody and the enforcement immunologic assay of preparation at mutant human growth hormone of the present invention to detect being described in more detail of mutant human growth hormone.
The purification of the mutant hGH that reorganization produces
In case confirmed the expression of recombinant mutant human growth hormone in the host cell of transfection,, host cell cultivated with appropriate scale for the purpose of purification of Recombinant body polypeptide.
The purification of the recombinant mutant hGH that in antibacterial, produces
Usually induce back (although express can be composition) in promoter, when the antibacterial that transforms produced mutant human growth hormone of the present invention in large quantities, protein can form the aggregation of indissoluble.There are some to be applicable to the method for protein purification inclusion body, for example, purification aggregation protein (to call inclusion body in the following text) be usually directed to by the cracking bacterial cell extract, separation and/or purification inclusion body, for example by in the non-ionic detergent buffer that contains about 100-150ug/ml lysozyme and 0.1%Nonidet P40, hatching.(Brinkman Instruments, Westbury NY) grind cell suspension can to use the Polytron grinder.In addition, can be with cell at ultrasonotomography on ice.Other method of decomposing bacteria is at Ausubel etc. and Sambrook and Russell, the middle description that sees above, and it will be apparent to those skilled in the art that.
Usually cell suspension is centrifugal, the resuspending in the buffer that is deposited in that contains inclusion body, the insoluble inclusion body of separating of this buffer still washs this inclusion body, for example 20mM Tris-HCl (pH7.2), 1mM EDTA, 150mM NaCl and 2% Triton-X 100 are a kind of non-ionic detergent.May need the repeated washing step to remove cell debris as much as possible.The residual grains of inclusion body can be in suitable buffer resuspending (for example, 20mM sodium phosphate, pH6.8,150mM NaCl).Other suitable buffer it will be apparent to those skilled in the art that.
Behind the washing step, by adding the dissolution with solvents inclusion body, this solvent is strong hydrogen acceptor and strong the hydrogen donor associating of the solvent of such character (or respectively have) simultaneously.Can be by making the protein renaturation that forms this inclusion body with compatible buffers dilution or dialysis.The suitable solvent includes, but are not limited to carbamide (from about 4M to about 8M), Methanamide (about at least 80% volume/volume) and guanidine hydrochloride (from about 4M to about 8M).Since protein possibility irreversible denaturation, and with immunogenicity and/or active disappearance, some can dissolve the proteinic solvent that forms aggregation, and for example SDS (sodium lauryl sulphate) and 70% formic acid can be unsuitable for this method.Although guanidine hydrochloride and similar agents are denaturants, this Denaturation is not irreversible, by removing (for example by dialysis) or diluting this denaturant renaturation can take place, and the immunity of destination protein matter and/or biologic activity are formed again.After the dissolving, can from other bacterioprotein, separate this protein by the standard isolation technics.Can be from further specifying of antibacterial inclusion body purification recombinant human growth hormone referring to Patra etc., ProteinExpression and Purification 18:182-190 (2000).
In addition, may be from the antibacterial pericentral siphon purification of Recombinant body polypeptide, for example mutant human growth hormone.When the pericentral siphon of recombinant protein input antibacterial, except other method well known by persons skilled in the art, all quality and grade parts that can be by cold osmotic shock separation of bacterial (for example referring to Ausubel etc., see above).In order to separate recombinant protein matter from pericentral siphon, centrifugal bacterial cell forms precipitation, and this is deposited in resuspending in the buffer that contains 20% sucrose.For cell lysis, centrifugal antibacterial, will be deposited in ice-cold 5mM MgSO
4Middle resuspending, and in ice bath, kept about 10 minutes.This cell suspension is centrifugal, and supernatant is moved into other containers preserve.Can from host protein, separate the recombinant protein that is present in supernatant by the well-known standard isolation technics of those skilled in the art.
The standard protein isolation technics that is used for purification
When the recombinant polypeptide, when mutant human growth hormone for example of the present invention expressed with soluble form in host cell, its purification can carry out according to standard protein purification method as described below.
Solubility fractionation
Often as initial step, if and protein mixture is very complicated, initial salt fractionation can be isolated the many unwanted host cell proteins matter protein of cell culture medium (or derive from) from for example mutant human growth hormone's of the present invention purpose recombinant protein.Preferred salt is ammonium sulfate, and ammonium sulfate is precipitating proteins by the amount that reduces the water in the protein mixture effectively, and protein then precipitates according to their dissolubility.Proteic hydrophobicity is big more, and it will may precipitate under lower ammonium sulfate concentrations more.A kind of typical step is to add saturated ammonium sulfate to make the ammonium sulfate concentrations of acquisition between 20-30% in protein solution.This will precipitate hydrophobic protein.Precipitation is abandoned (unless destination protein is hydrophobic), and adding ammonium sulfate reaches the known concentration that can precipitate destination protein in supernatant.Then resolution of precipitate in buffer, if necessary by the dialysis or percolation remove excessive salt.Other method of the solubility of dependent protein matter, for example the cold ethanol precipitation method is well-known to those skilled in the art, can be used for the protein mixture of separate complex.
Difference in size is filtered
Based on the molecular weight that calculates, can pass through the Ultrafiltration of the film (for example, Amicon or Millipore film) of different pore sizes and separate albumen with big and reduced size.As the first step, by having than destination protein, the film of the weight shutoff pore size that for example mutant human growth hormone's molecular weight is lower carries out ultrafiltration protein mixture.Then with the ultrafiltrate that has greater than the membrane ultrafiltration back of the molecule cutoff of the molecular weight of destination protein.Recombinant protein will enter filtrate by this film, then according to following method chromatography filtrate.
Column chromatography
Can also separate destination protein matter (mutant human growth hormone for example of the present invention) according to their size, clean surface charge, hydrophobicity or aglucon affinity.In addition, human growth hormone's antibody can be connected with base for post matter, and the human growth hormone is by immune purification.All these methods are well known in the prior art.
Can use with the equipment that is used for from many different manufacturers (for example Pharmacia Biotech) with scale arbitrarily the apparent chromatographic technique of technical staff and to carry out.
Detect the immunoassay that mutant hGH expresses
In order to confirm recombinant mutant human growth hormone's generation, immunoassay can be used for detecting polypeptide expression at sample, and immunoassay also can be used for quantizing the expression of recombinant hormone.Antibody at the mutant human growth hormone is to realize that these immunoassays are necessary.
Production at the antibody of mutant hGH
Produce the polyclonal and monoclonal antibody method of reacting specifically with the purpose immunogen to those skilled in the art be known (for example referring to, Coligan, CurrentProtocols in Immunology Wiley/Greene, NY, 1991; Harlow and Lane, Antibodies:A Laboratory Manual Cold Spring Harbor Press, NY, 1989; Stites etc. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and the list of references of wherein quoting; Goding, Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, NY, 1986; And Kohler and Milstein, Nature 256:495-497,1975).Above-mentioned technology comprise by from phage or similarly the recombinant antibody storehouse the carrier select antibody Antibody Preparation (referring to, Huse etc., Science 246:1275-1281,1989; And Ward etc., Nature 341:544-546,1989).
Contain the antiserum with required specific antibody in order to produce, desired polypeptides (mutant human growth hormone for example of the present invention) or its antigenicity fragment can be used for the suitable animal of immunity, for example mice, rabbit or primate.Can use standard adjuvant, for example Freund adjuvant according to the immune step of standard.In addition, the synthetic antigenic peptide from specific polypeptide can be connected with carrier protein also subsequently as immunogen.
By obtaining chemical examination blood and measuring the antigenic reactive titre of purpose is come the immunoreation of monitor animal to the immunogen goods.When having obtained antibody, collect blood and prepare antiserum from animal to the suitably high titre of antigen.Then can implement sero-fast further classification with enrichment to the antibody of antigen specific reaction and can carry out further antibody purification, referring to Harlow and Lane, see above, and the general remark of protein purification provided above.
Use and be multiple technologies acquisition monoclonal antibody well known to those skilled in the art.Usually splenocyte of the animal of the required antigen immune of using by oneself and myeloma cell are merged and immortalization (referring to Kohler and Milstein, Eur J.Immunol.6:511-519,1976).Other method of immortalization comprises for example uses Epstein-Barr virus, oncogene or retrovirus to transform, or well-known other method in the prior art.The specificity that examination needs from having of the clone of single immortalized cells and to the production of antibodies of antigenic affinity can improve the output of the monoclonal antibody of this cells produce by multiple technologies, comprises the peritoneal cavity that is injected into vertebrate host.
In addition, by the conventional method screening human B cell cDNA library of describing according to (as mentioned) such as Huse, the nucleotide sequence of the specific antibody that identification code needs or the binding fragment of this antibody, thereby can the recombinant production monoclonal antibody, the rule of the recombinant polypeptide production of above-mentioned discussion and method are applicable to by recombination method produces antibody.
When needs, can test the antibody that to discern mutant human growth hormone of the present invention specifically cross reactivity, thereby distinguish mutually with the antibody of wild-type protein to the wild type human growth hormone.For example, the antiserum that obtains from the animal with mutant human growth hormone immunity can only be discerned the mutant human growth hormone by the antiserum part of pillar by having fixed wild type human growth hormone's pillar, and nonrecognition wild type human growth hormone.The exclusiveness of only discerning mutant rather than wild type human growth hormone of monoclonal antibody that similarly, can also examination mutant human growth hormone.Similarly, monoclonal antibody that can also examination mutant human growth hormone only discern mutant and nonrecognition wild type human growth hormone's specificity.
Only discern mutant human growth hormone of the present invention specifically and nonrecognition wild type human growth hormone's polyclone or monoclonal antibody can be used for segregation mutant albumen from wild-type protein, for example hatch by polyclone or monoclonal antibody that sample is special with being fixed on mutant human growth hormone on the solid carrier.
Detect the immunoassay that mutant hGH expresses
In case can obtain mutant human growth hormone's of the present invention specific antibody, can provide the method for immunity of qualitative and quantitative result to detect the quantity of polypeptide in sample (for example lysate) to those skilled in the art by various.To the summary of general immunologic and method of immunity referring to, for example Stites sees above; U.S. the patent No. 4,366, and 241; 4,376,110; 4,517,288; With 4,837,168.
The labelling of immunoassay
Immunoassay usually utilization and antibody and target protein form combine complex specifically in conjunction with and the labelled reagent of labelling.Labelled reagent itself can be a part that contains antibody/target protein complex, maybe may be third part, for example with bonded specifically another antibody of antibody/target protein complex.Can pass through spectroscope, photochemistry, biochemistry, immunochemistry, electricity, optics or chemical method certification mark.Example includes, but are not limited to magnetic bead (Dynabeads for example
TM), fluorescent dye (for example Fluorescein isothiocyanate, texas Red, rhodamine or the like), radioactive label (for example
3H,
125I,
35S,
14C or
32P), enzyme (for example being generally used for horseradish peroxidase, alkali phosphatase of ELISA or the like), and colorimetric labelling for example gold colloidal or coloured glass or plastics (for example polystyrene, polypropylene, latex or the like) pearl.
Sometimes labelled reagent is the second antibody that has detectable labelling.In addition, second antibody can not have labelling, but it can be subsequently by the specificity of labelling the 3rd antibodies at the antibody of the species in second antibody source.Second antibody can be modified with detectable part, the biological example element, and the 3rd labelled molecule is the combination with it specifically of enzyme mark streptavidin for example.
Other is the albumen of binding domain-immunoglobulin constant region specifically, and for example A albumen or G albumen also can be used as labelled reagent.These protein are normal components of streptococcus cell wall.They demonstrate with from the strong non-immunogenic reactivity of the constant region of the immunoglobulin of various species (usually referring to, Kronval, etc., J.Immunol., 111:1401-1406 (1973); And Akerstrom, etc., J.Immunol., 135:2589-2542 (1985)).
The immunoassay form
Detection can be emulative or noncompetitive from the immunoassay of the interested target protein (for example mutant human growth hormone) of sample.Non-competitive immunoassay is the analysis of directly measuring the quantity of the target protein of capturing.For example, in a kind of preferred " sandwich " analyzed, the specific antibody of target protein can directly combine with the solid matrix of sessile antibody.Capture the target protein in the test specimen then.Fixed antibody like this/target protein complex is then by labelled reagent, for example aforesaid the second or the 3rd antibodies that has label.
In competitive assay, replace the quantity of (ectogenic) target protein that (or competition is fallen) add and measure the quantity of target protein indirectly from the antibody that is specific to described target protein by measuring by the target protein that is present in the sample, in the representative instance of a this analysis, the ectogenic target protein of sessile antibody and labelling.Owing to be inversely proportional to the concentration that is present in the target protein in the sample with the quantity of the ectogenic target protein of antibodies, thereby therefore based on determining the sample proteic level that hits with the quantity of the fixed exogenous target protein of antibodies.
Sometimes, adopt the existence of mutant human growth hormone in western hybridization (the immune marking) detection and the quantitative sample.This technology generally includes by gel electrophoresis according to molecular weight sample separation albumen, isolating albumen is transferred to suitable solid carrier (for example nitrocellulose filter, nylon membrane or deutero-nylon membrane), and sample with hatch with the bonded antibody of target protein specifically.These antibody directly labelling maybe can use specifically the antibody (for example goat anti-mouse antibody of labelling) with the labelling of mutant human growth hormone's antibodies to detect subsequently.
Other analytical form comprises liposome immunoassay (LIA), its use is designed to and can and discharges the reagent of sealing or the liposome of labelling in conjunction with specific molecular (for example antibody), the chemicals that detect to discharge according to standard technique is (referring to Monroe etc. then, Amer Clin.Prod.Rev., 5:34-41 (1986)).
Glycosylation and the sugar of mutant hGH are puted together
Enzymatic glycosylation and sugar are puted together
External modification is that a kind of remedying depends on the attracting strategy that the manipulation expression system is controlled the defective of glycosylated method after the expression of peptide; Comprise the change of glycan structures or import polysaccharide in new site.Can utilize the enzyme that shifts saccharide donor part widely, make can external enzymatic synthetic the have glycosylation pattern of custom design and the glycoconjugate of glycosyl structure, for example referring to, the U.S. patent No. 5,876,980; 6,030,815; 5,728,554; 5,922,577; And disclosed patent application WO 98/31826; WO 01/88117; WO 03/031464; WO03/046150; WO 03/045980; WO 03/093448; WO 04/009838; US2002/142370; US2003/040037; US2003/180835; US2004/063911; US2003/207406; And US2003/124645.
The invention provides the method for the glycosylated and nonglycosylated mutant human growth hormone's of preparation conjugate, this mutant human growth hormone has the non-existent new glycosylation site of corresponding wild type hGH.Above-mentioned puts together in the suitable sugared unit that can directly occur in glycosylated mutant hGH, or takes place after removing (promptly removing) unwanted arbitrarily sugared unit.Put together between the variety classes that is formed on peptide and for example water-soluble polymer, therapeutic part, diagnosis part, targeting moiety etc.Also provide the conjugate that comprises two or more peptides that connect by linking arm, promptly multi-functional conjugate.Multi-functional conjugate of the present invention can comprise the same peptide or the various set that the peptide of different structures and/or character is arranged of two or more copies.
Conjugate of the present invention forms by modifying sugared the connection with glycosylated or nonglycosylated peptide enzymatic.Become in the time of between the modification group on modifying sugar insertion peptide and sugar alleged here " complete glycosyl linking group ".Use the selectivity of the acumen of enzyme, glycosyl transferase for example, this method provides the peptide that has required group at one or more specific sites.Thereby, according to the present invention, directly is connected modifying sugar, perhaps the sugared carbohydrate part that is attached to glycopeptide of modification with the site selected on the peptide chain.Wherein modify sugar is connected with the glycopeptide carbohydrate simultaneously with the direct peptide that is connected with the amino acid residue of peptide backbone also within the scope of the invention.
Opposite with enzymatic peptide Processing Strategies with known chemistry, method of the present invention makes peptide and glycopeptide with the substantially the same pattern of deriving combined becomes possibility; Be used for enzyme of the present invention and usually the combination of the amino acid residue of particular amino acid residue or peptide had selectivity.This method also is practical to the peptide of modification and the large-scale production of glycopeptide.Therefore, method of the present invention provides the practical way of mass preparation of glycopeptide of the pattern of deriving of the unification with preliminary election.This method is particularly suitable for the modification of therapeutic peptide, includes but not limited to cell (for example mammalian cell, insect cell, plant cell, fungal cell, yeast cells or prokaryotic cell) or transgenic plant or the glycosylated by halves glycopeptide of animal production period at cell culture.
Method of the present invention provides the conjugate of the glycosylated and non-glycosylated peptide of the therapeutic half-life that has raising owing to the clearance rate that reduces or the immunity of reduction or the picked-up of reticuloendothelial system (RES) etc. in addition.In addition, method of the present invention provides the mode of the antigenic determinant on the shielding peptide, and therefore reduction or elimination are at the host immune response of this peptide.The selectivity connection of target reagent can also be used to making the peptide targeting extremely to specific specific tissue of described particular target reagent or cell surface receptor.
Conjugate
First aspect the invention provides at the modification group of selecting and has conjugate between the hGH mutant peptide of non-existent glycosylation site in the wild type peptide.Modification group can be connected with mutant glycosylation site or the site that is present in wild type peptide.
Connection between peptide and the modification group comprises the glycosyl linking group between the part of inserting peptide and selection.As discussing herein, thus the part of selection come down to be connected in sugared unit produces can be by any kind of the modification sugar of suitable transferring enzyme identification, this transferring enzyme will be modified and sugaredly be connected with peptide.When the saccharic composition of modifying sugar inserts between the part of peptide and selection, become " glycosyl linking group ", for example " complete glycosyl linking group ".The glycosyl linking group is made up of any monosaccharide or oligosaccharide, and after modifying with modification group, it is to add the substrate of modifying sugared enzyme to the aminoacid of peptide or glycosyl residue.
This glycosyl linking group can be maybe can be included in to add the sugar moieties that degradability is modified during the modification group.For example, the saccharide residue that the glycosyl linking group produces in the time of can coming free complete glycosyloxyization to be degraded to corresponding aldehyde for example by the effect of inclined to one side periodates, becomes Schiff's base with suitable amine subsequently, is reduced into corresponding amine then.
Conjugate of the present invention meets array structure down usually:
Wherein symbol a, b, c, d and s represent the positive integer of non-zero; T is 0 or positive integer." reagent " is therapeutic agent, bioactive agents, detectable labelling, water-soluble portion (for example PEG, m-PEG, PPG and m-PPG) or the like." reagent " can be a kind of peptide, for example enzyme, antibody, antigen or the like.This connexon can be the linking group of any broad array, and is as described below.In addition, connexon can be singly-bound or " zero level connexon ".
In a kind of exemplary specific embodiment, the modification group of selection is a kind of water-soluble polymer, for example m-PEG.Water-soluble polymer is covalently bound via glycosyl linking group and peptide.This glycosyl linking group is covalently attached to the amino acid residue or the glycosyl residue of peptide.The present invention provides the conjugate of wherein using glycosyl linking group modified amino acid residue and glycosyl residue in addition.
A kind of exemplary water-soluble polymer is poly-(ethylene glycol), for example methoxyl group poly-(ethylene glycol).Be used for poly-(ethylene glycol) of the present invention and be not limited to any particular form or molecular weight ranges.Poly-(ethylene glycol) molecular weight preferably between 500 and 100,000, preferably adopts 500-60, and 000 molecular weight is preferably 1, and 000-40 000, is more preferably molecular weight from about 5,000 to about 40,000.
Poly-(ethylene glycol) is a kind of PEG of branch that has more than the peg moiety of a connection in another embodiment.The example of the PEG of branch is described in following document: the U.S. patent No. 5,932,462; U.S. the patent No. 5,342, and 940; U.S. the patent No. 5,643, and 575; U.S. the patent No. 5,919, and 455; U.S. the patent No. 6,113, and 906; U.S. the patent No. 5,183, and 660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5:283-288 (1994); And Yamasaki etc., Agric.Biol.Chem., 52:2125-2127,1998.Poly-(ethylene glycol) molecular weight of each PEG of branch is 5 in a preferred scheme, 000-20,000.
Add the conjugate of glycosyl linking group formation except that providing, the invention provides the highly homogeneous conjugate of substitute mode by enzymatic.Use method of the present invention, may form the peptide conjugate of modifying sugar moieties aminoacid identical or glycosyl residue connection in the colony of all conjugates of the present invention basically with a plurality of structures.Therefore, second aspect the invention provides the peptide conjugate with water-soluble polymer partial mass, and this water-soluble polymer is partly by complete glycosyl linking group and peptide covalent bond.In a preferred conjugate of the present invention, each member basically of this colony is connected with the glycosyl residue of peptide via the glycosyl linking group, and each glycosyl residue of the peptide of glycosyl linking group connection all has same structure.
A kind of peptide conjugate that has by the covalently bound with it water-soluble polymer partial mass of glycosyl linking group is provided in addition.In a preferred scheme, each member basically of this water-soluble polymer partial mass is connected with the amino acid residue of peptide via the glycosyl linking group, and each has the amino acid residue that the glycosyl linking group connects and all has same structure.
The present invention provides the analog of above-mentioned conjugate in addition, and wherein peptide is puted together via complete glycosyl linking group and therapeutic part, diagnosis part, targeting moiety, toxin moiety.Each above-mentioned part can be micromolecule, natural polymer (for example polypeptide) or synthetic polymer.
In a kind of exemplary specific embodiment, the mutant human growth hormone is connected with transferrin via bifunctional connexon, and this connexon comprises complete glycosyl linking group (scheme 1) at the end points of each peg moiety.For example, an end points of PEG connexon is with complete sialic acid connexon functionalization, and this sialic acid connexon is connected with transferrin, and another end points is with complete GalNAc connexon functionalization, and this GalNAc connexon is connected with mutant hGH.
Conjugate of the present invention can comprise unit price or polyvalent intact glycosyl linking group (for example feeler structure).Therefore, conjugate of the present invention comprises two kinds of types, and wherein the part of Xuan Zeing is connected with peptide via unit price glycosyl linking group.Wherein the conjugate that is connected with peptide via polyvalent linking group more than the part of a selection is also included within the scope of the present invention.
In another specific embodiment, the invention provides owing to optionally be positioned at the conjugate of particular organization as the existence of the targeting agent of conjugate component.In a kind of exemplary specific embodiment, targeting agent is a kind of albumen.Exemplary albumen comprises transferrin (brain; the blood storehouse); HS-glycoprotein (bone; brain; the blood storehouse); antibody (brain; has the special antigenic tissue of antibody; the blood storehouse); labile factor-XII (injured tissues; grumeleuse; cancer; the blood storehouse); serum albumin, for example alpha-acid glycoprotein; myosin; α-fetus albumen (brain; the blood storehouse); beta 2-glycoprotein (liver; atherosclerotic plaque; brain; the blood storehouse); G-CSF; GM-CSF; M-CSF and EPO (immunostimulation; cancer; the blood storehouse; erythrocyte excessively generates; neuroprotective); albumin (half-life increase) and lipoprotein E.
Method
Except that the above-mentioned conjugate that discusses, the invention provides the method for preparation these and other conjugate.Therefore, on the other hand, the invention provides a kind of method that between part of selecting and peptide, forms covalent conjugates.In addition, the invention provides particular organization or the regional method that conjugate of the present invention is oriented to body.And, the invention provides a kind of by giving the method for preventing, treat or improve morbid state with conjugate of the present invention to the individuality that the individual of onset risk is arranged or have a disease.
In the exemplary specific embodiment, conjugate forms between water-soluble polymer, therapeutic part, targeting moiety or biomolecule and glycosylated or nonglycosylated peptide.Polymer, therapeutic part or biomolecule combine via the insertion centre and with peptide and the covalently bound intact glycosyl linking group of modification group (for example water-soluble polymer) with peptide.This method comprises makes peptide contact with the mixture of glycosyl transferase with containing modification sugar, and the substrate of described glycosyl transferase is described modification sugar.Be reflected to make to modify under the condition that enough forms covalent bond between sugar and the peptide and carry out.The sugar moieties of modifying sugar is preferably from nucleotide sugar, activation sugar with neither select the also inactive sugar of nucleotide sugar.
Receptor peptide (glycosylated or nonglycosylated) is synthetic from the beginning generally, or recombinant expressed in prokaryotic cell (for example bacterial cell, as escherichia coli) or express in eukaryotic cell such as mammal, yeast, insecticide, fungus or plant cell.Peptide can be full-length proteins or fragment.Peptide can be wild type or mutant peptide in addition.In a kind of exemplary specific embodiment, peptide comprises the sudden change that adds one or more total glycosylation sites in peptide sequence.
Method of the present invention also provides the modification of the incomplete glycosylated peptide of recombinant production.The glycoprotein of many recombinant production is not exclusively glycosylated, exposes to have the immunogenic carbohydrate residue that undesirable character is for example discerned by RES.Adopt to modify sugar in the method for the invention, peptide can be simultaneously further glycosylation and with for example water-soluble polymer, therapeutic agent or the like derivatization.The sugar moieties of modifying sugar can be with abundant glycosylated peptide in the residue suitably puted together of receptor, or another has the sugar moieties of the character of catering to the need.
The peptide of modifying with method of the present invention can be synthetic or wild type peptide, maybe can be the mutant peptide of being produced by methods known in the art such as direct mutagenesis.The glycosylation of peptide generally is that N-connects or O-connects.It is to modify sugar to be connected with asparagine residue side chain that a kind of exemplary N connects.Tripeptide sequence agedoite-X-serine and agedoite-X-threonine, wherein X is the arbitrary amino acid except that proline, is the recognition sequence that the carbohydrate part is connected with agedoite side chain enzymatic.Therefore, any one existence of these tripeptide sequences produces potential glycosylation site in the polypeptide.O-connects the hydroxyl that glycosylation refers to monosaccharide (for example N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose or xylose) and hydroxy-amino-acid side chain, preferred serine or threonine connect, but also can use 5-hydroxyproline or 5-oxylysine.
Adding glycosylation site to peptide or other structure makes it contain one or more glycosylation sites and finish expediently by changing aminoacid sequence.This interpolation also can have the kind of OH group by introduce one or more (O-connects glycosylation site) in peptide sequence, preferably serine or threonine residues and finish.This interpolation can be finished by whole chemosynthesis of sudden change or peptide.The peptide ammino acid sequence preference is by changing at dna level, suddenlys change by the DNA base at the encoded peptide of preliminary election especially, makes the codon of generation translate into the aminoacid that needs.Dna mutation preferably uses methods known in the art to finish.
In a kind of exemplary specific embodiment, glycosylation site adds by resetting polynucleotide.The polynucleotide of coding candidate peptide can be adjusted with the DNA rearrangement method.It is a kind of circulation reorganization and mutation method that DNA resets, and is undertaken by random fragmentation related gene storehouse, by the method for polymerase chain reaction sample fragment is ressembled then.For example referring to, Stemmer, Proc.Natl.Acad.Sci.USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); And the U.S. patent No. 5,605,793,5,837,458,5,830,721 and 5,811,238.
The present invention provides the mode of adding the glycosyl residue of (or removing) one or more selections to peptide in addition, puts together to the glycosyl residue of at least one selection of peptide thereafter and modifies sugar.This specific embodiment can be applicable to, and does not for example have glycosyl residue on the peptide or when not existing with the amount that requires, need connect the situation of modifying sugar to the glycosyl residue of selecting.Therefore, to peptide in conjunction with before modifying sugar, by enzymatic or chemical coupling put together the glycosyl residue of selection to peptide.In another embodiment, before connect modifying sugar by remove the glycosylation pattern that the carbohydrate residue changes glycopeptide from glycopeptide.For example referring to WO 98/31826.
Add or remove any carbohydrate that exists on the glycopeptide and partly finish with chemistry or Enzymology method.The chemistry deglycosylation is preferably finished by the chemical compound that polypeptide variants is exposed to chemical compound trifluoromethanesulfonic acid or equivalence.This processing causes great majority or whole sugared cracking of except that connecting sugar (N-acetyl-glucosamine or N-acetylgalactosamine), and stays complete peptide.Hakimuddin etc., Arch.Biochem.Biophys.259:52 (1987) and Edge etc., Anal.Biochem.118:131 (1981) has described chemical deglycosylation.On the polypeptide variants enzyme of carbohydrate part urge cracking can by utilize various inscribes-and circumscribed-glycosidase finish, as Thotakura etc., Meth.Enzymol.138:350 (1987) is described.
The chemistry of glycosyl part adds and is undertaken by any recognized techniques method.The enzymatic of sugar moieties adds the improving one's methods of method that preferred use illustrates herein to be finished, and replaces with natural glycosyl unit that to be used for modifications of the present invention sugared.Other the method for adding sugar moieties is in the U.S. patent No. 5,876,980,6,030 815,5,728, and is open in 554 and 5,922,577.
The exemplary junction point of the glycosyl residue of selecting including, but not limited to: (a) N-connects glycosylation and is connected glycosylated total site with O-; (b) as the terminal saccharide part of glycosyl transferase receptor; (c) arginine, agedoite and histidine; (d) free carboxyl group; (e) sulfydryl of free sulfydryl such as cysteine; (f) hydroxyl of free hydroxyl such as serine, threonine or hydroxyproline; (g) those residues of aromatic residue such as phenylalanine, tyrosine or tryptophan; Or (h) amide groups of glutamine.Can be used for exemplary method of the present invention at WO 87/05330, open day Sep.11,1987, and Aplin and Wriston, describe among the CRCCRIT.REV.BIOCHEM., pp.259-306 (1981).
In a specific embodiment, the invention provides a kind of method that connects hGH and one or more peptides by linking group.Linking group is adoptable arbitrarily structure, can select from straight chain and branched structure, and preferably the end of each connexon that is connected with peptide comprises modification sugar (being the intact glycosyl linking group at initial stage).
In a kind of exemplary method of the present invention, two peptides are coupled together via the connexon part that comprises the PEG connexon.Structure conforms to the general structure that above-mentioned picture is represented.Comprise two complete glycosyl linking groups (being s+t=1) as structure of the present invention described herein.Focus on the PEG connexon that comprises two glycosyl groups is for purpose, the not character of the connexon arm that adopts in the specific embodiment that should be construed as limiting the invention clearly.
Therefore, with the first glycosyl unit at first end points, with the second glycosyl unit at the second endpoint functionality peg moiety.The substrate of the transferring enzyme that the first and second glycosyl units are preferably different allows first to be connected with the first and second glycosyl unit right angles respectively with second peptide.In practice, (glycosyl)
1-PEG-(glycosyl)
2Connexon contacts with first transferring enzyme that with the first glycosyl unit is substrate with first peptide, thereby forms (peptide)
1-(glycosyl)
1-PEG-(glycosyl)
2From reactant mixture, optionally remove glycosyl transferase and/or unreacted peptide then.Second peptide with the second glycosyl unit is second transferring enzyme adding (peptide) of substrate
1-(glycosyl)
1-PEG-(glycosyl)
2Conjugate forms (peptide)
1-(glycosyl)
1-PEG-(glycosyl)
2-(peptide)
2It will be appreciated by those skilled in the art that above-mentioned method also applicable between more than two peptides, forming conjugate, for example by utilizing the PEG of branch, dendritic, poly-(aminoacid), polysaccharide material or the like.
In a kind of exemplary specific embodiment, the mutant human growth hormone is connected with transferrin via bifunctional connexon, and this connexon comprises complete glycosyl linking group (scheme 1) at the end of each peg moiety.Because the bigger molecular size of conjugate, hGH conjugate have the half-life in the body that increases than independent hGH.In addition, hGH and transferrin puts together that to make conjugate optionally be target with the brain.For example, of PEG connexon is terminal with CMP sialic acid functionalization, and another is terminal with UDP GalNAc functionalization.Under the situation that the GalNAc transferring enzyme exists, this connexon combines with hGH, causes the GalNAc of connexon arm to be connected with serine and/or threonine residues on the hGH.
Aforesaid operation can be carried out any period how to be finished, and is not limited between two peptides with single connexon formation conjugate.In addition, it will be appreciated by those skilled in the art that with peptide the reaction of the intact glycosyl linking group functionalization of PEG (or other) connexon end can taken place simultaneously with in-reaction vessel, or they can in a step-wise fashion carry out.When reaction is when in a step-wise fashion carrying out, the conjugate of producing in each step is purification from one or more reactive components (for example enzyme, peptide) alternatively.
Another exemplary specific embodiment is set forth in scheme 2, and scheme 2 shows albumen that a kind of preparation will select for example human growth hormone's targeting bone and the method that increases the proteic circulating half-life of selecting.
Wherein G is the glycosyl residue on activation sugar moieties (for example ribotide), and it is transformed into the intact glycosyl connexon in conjugate.When s greater than 0 the time, L is the glycosyl linking group as GalNAc or GalNAc-Gal.
Utilize the reactive derivatives of PEG (or other connexon) to add one or more peptide moieties within the scope of the invention to connexon.The invention is not restricted to the character of reactive PEG analog, the activated derivatives of many Polyethylene Glycol can be in commercial and acquisition in the literature.Select and in case of necessity a kind of suitable activated PEG derivant of synthetic be used for substrate of the present invention fully in technical staff's limit of power with preparation.Referring to, people CancerBiochem.Biophys. such as Abuchowski, 7:175-186 (1984); Abuchowski etc., J.Biol.Chem., 252:3582-3586 (1977); Jackson etc., Anal.Biochem., 165:114-127 (1987); Koide etc., Biochem Biophys.Res.Commun., 111:659-667 (1983)), tresylate (Nilsson etc., Methods Enzymol., 104:56-69 (1984); People such as Delgado, Biotechnol.Appl.Biochem., 12:119-128 (1990)); The deutero-active ester of N-hydroxyl succinyl (Buckmann etc., Makromol.Chem., 182:1379-1384 (1981); Joppich etc., Makromol.Chem., 180:1381-1384 (1979); Abuchowski etc., Cancer Biochem.Biophys., 7:175-186 (1984); Proc.Natl.Acad.Sci.U.S.A. such as Katre, 84:1487-1491 (1987); Kitamura etc., Cancer Res., 51:4310-4315 (1991); Boccu etc., Z.Naturforsch., 38C:94-99 (1983), carbonic ester (Zalipsky etc., POLY (ETHYLENE GLYCOL) CHEMISTRY:BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, Harris, Ed., PlenumPress, New York, 1992, pp.347-370; Zalipsky etc., Biotechnol.Appl.Biochem., 15:100-114 (1992); Veronese etc., Appl.Biochem.Biotech., 11:141-152 (1985)), imidazole radicals formic acid (Beauchamp etc., Anal.Biochem., 131:25-33 (1983); Berger etc., Blood, 71:1641-1647 (1988)), 4-dithio pyridine (Woghiren etc., Bioconjugate Chem., 4.314-318 (1993)), Carbimide. (Byun etc., ASAIO Journal, M649-M-653 (1992)) and epoxide (U.S.Pat.No.4,806,595, authorize Noishiki etc., (1989).Other linking group comprises that the urethane between amino group and the activated PEG connects, referring to Veronese, etc., Appl.Biochem.Biotechnol., 11:141-152 (1985).
In another exemplary specific embodiment, the invention provides the method for a kind of blood circulation half-life of the peptide that prolongs selection, make peptide targeting blood storehouse in fact, by enough big or small synthetic or natural polymer (for example albumin) is connected with peptide to postpone the filtration of albumen by glomerule, referring to scheme 3.This specific embodiment of the present invention illustrates that in scheme 3 wherein hGH is connected with the combination of enzymatically modifying via PEG connexon utilization chemistry with albumin.
Scheme 3
Therefore shown in scheme 3, with reactive PEG derivant for example X-PEG-(cmp sialic acid) change albuminous residue (for example amino acid side chain), wherein X is a kind of activated group (for example Acibenzolar, isothiocyanate or the like).The PEG derivant is contacted with the hGH combination and with the transferring enzyme that with the cmp sialic acid is substrate.In the illustrative specific embodiment of another one, the reaction of the N-hydroxy-succinamide ester of the ε-amine of lysine and PEG connexon is to form the albumin conjugate.Suitable residue on the cmp sialic acid of connexon and the hGH for example Gal or GalNAc enzymatic is connected, thereby forms conjugate.The technical staff will understand the reactant that above-mentioned method is not limited to elaboration, and this method can form the conjugate that comprises more than two protein parts, for example have ramose connexon more than two ends by utilization.
Modify sugar
The glycosyl donor kind of modifying (" modifying sugar ") preferably from the ribotide of modifying, activatory modification sugar and both non-nucleotide also select the modification sugar of inactive monosaccharide.Can use method of the present invention to add the carbohydrate structure that requires arbitrarily to peptide.Usually, this structure is a monosaccharide, but the present invention is not limited to the monosaccharide that utilizes modification; Also can adopt oligosaccharide and polysaccharide.
Modification group and sugar moieties are connected by enzymatic mode, chemical method or its, modify sugar thereby produce.This sugar is replacing for modifying any site that part connects, yet still allows sugar to play a role as the substrate that is used to connect the enzyme of modifying sugar and peptide.In a preferred scheme, when sugar is sialic acid, replace sialic acid in pyruvyl side chain site 9 or in sialic acid on the site 5 of common acetylizad amine moiety with modification group.
In some the specific embodiment of the present invention, use the ribotide of modifying to add and modify sugar to peptide.Be used for exemplary modification type ribotide of the present invention and comprise nucleotide single, double or triphosphoric acid or its analog.In a preferred scheme, the ribotide of modification is selected from UDP-glucosides, CMP-glucosides or GDP-glucosides.Be more preferably, the ribotide of modification is selected from UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glycosamine, GDP-mannose, GDP-fucose, cmp sialic acid or CMP-NeuAc.The N-acetamide derivative of ribotide also can be used in the method for the present invention.
The present invention also provides the method for using the peptide of modifying sugared synthetic modification, for example galactose of Xiu Shiing, fucose, GalNAc and sialic acid.When adopting the sialic acid of modifying, saliva based transferase or commentaries on classics sialidase (only being used for α 2, the sialic acid that 3-connects) can be used for these methods.
In other the specific embodiment, modifying sugar is a kind of activatory sugar.Being used for activatory modification sugar of the present invention generally is glucosides, and the manual change is for containing activatory leaving group.The term of Cai Yonging " activatory leaving group " refers in the nucleophilic substitution that enzyme is regulated easily by metathetical part herein.A lot of activatory sugar known in the art.For example referring to Vocadlo etc., In CARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol.2, Ed. such as Ernst, Wiley-VCH Verlag:Weinheim, Germany, 2000; Kodama etc., Tetrahedron Lett.34:6419 (1993); Lougheed, etc., J.Biol.Chem.274:37717 (1999)).
The example of activated group (leaving group) comprises fluoro, chloro, bromo, tosylate, methanesulfonates, triflate (triflate ester) or the like.Activatory leaving group preferred for the present invention is on those space structures glucosides to be shifted the activation leaving group that does not have remarkable obstruction to the receptor enzymatic.Therefore, the preferred version of activation glycosides derivatives comprises glycosyl fluoride and glycosyl methanesulfonates, and more preferably glycosyl fluoride.In the glycosyl fluoride, most preferably α-galactose fluoride, α-mannose fluoride, alpha-glucosyl fluoride, α-fucosido fluoride, α-xylosyl fluoride, α-sialic acid fluoride, α-N-acetyl glucosamine fluoride, α-N-acetyl group galactosamine fluoride, beta galactose fluoride, β-mannose fluoride, β-glucityl fluoride, β-fucosido fluoride, β-xylosyl fluoride, β-sialic acid fluoride, β-N-acetyl glucosamine fluoride and β-N-acetyl group galactosamine fluoride.
As example, the glycosyl fluoride can be handled with the HF/ pyridine then and prepare from free sugar by at first making sugared acetylation.This has produced (acetylizad) glycosyl fluoride anomer (being the alpha-glycosyl fluoride) of the most stable protection on the thermodynamics.More unsettled if desired anomer (being β-glycosyl fluoride) can acetylizad sugar produces different bromide or chloride prepares this anomer by changing with HBr/HOAc or with HCI.This intermedium with as the fluoride salt of Argentous fluoride reaction generation glycosyl fluoride.Acetylizad glycosyl fluoride can by with methanol in gentle (catalytic) alkali (for example NaOMe/MeOH) reaction go protection.Available many glycosyl fluorides on the market in addition.
Other activatory glycosyl derivatives can use conventional process preparation well known by persons skilled in the art, and for example, the glycosyl methanesulfonates can be by handling the sugar of the hemiacetal form of henzylate fully with mesyl chloride, and catalytic hydrogenation is removed benzyl and prepared then.
In another exemplary specific embodiment, modifying sugar is a kind of oligosaccharide with feeler structure.In a preferred scheme, the end of one or more feelers has the modification part.When being connected with the oligosaccharide with feeler structure more than a modification part, oligosaccharide can be used for " amplification " and modifies part; Each oligosaccharide unit that is connected with peptide is connected the modification group of a plurality of copies with peptide.The general structure of the typical chelate of the present invention of setting forth among the above-mentioned figure comprises and comes from the multivalence kind of utilizing the feeler structure to prepare conjugate of the present invention.Many antenniferous sugared structures known in the art, this method can be implemented with them ad lib.
Exemplary modification group is discussed hereinafter.Can give the ability of the one or more desirable character of polypeptide and select modification group according to them, exemplary character including, but not limited to the bio distribution of the pharmacodynamics of enhanced pharmacokinetics, raising, improvement, the multivalence kind be provided, improve water solublity, raising or reduce lipotropy and tissue target to.
Water-soluble polymer
By with polar molecule as containing amine, ester, hydroxyl and polyhydric molecule improve peptide in conjunction with the peptide of selecting hydrophilic.Representational example is including, but not limited to polylysine, polymine and polyethers, and for example poly-(ethylene glycol), m-poly-(ethylene glycol), poly-(propylene glycol), m-poly-(propylene glycol) and other O-alkyl gather (aklylene glycol) part.Preferred water-soluble polymer does not fluoresce substantially, or emission fluorescence in a small amount, and is unsuitable for the fluorescent labeling in performing an analysis.And usually preferred employing is not the polymer of naturally occurring sugar.A kind of this preferred exceptional case is the naturally occurring sugar that utilizes by another entity (for example, poly-(ethylene glycol), poly-(propylene glycol), biomolecule, therapeutic part, diagnosis part or the like) covalent attachment.In another exemplary specific embodiment, the therapeutic sugar moieties is connected with the connexon arm, via method of the present invention sugar-connexon arm box is connected with peptide subsequently.
Activation method and the chemical action of water-soluble polymer and sugar and the method that sugar and polymer are connected with different kind described in the literature.Usually the method for the activated polymer that adopts comprise with activation functional groups such as Bromine cyanide., periodic acid, glutaraldehyde, diepoxide (biepoxides), chloropropylene oxide, divinyl sulfone, carbodiimide, sulfuryl halide, three chlorotriazines (referring to, R.F.Taylor, (1991), PROTEIN IMMOBILISATION.FUNDAMENTALS ANDAPPLICATIONS, MarcelDekker, N.Y.; S.S.Wong, (1992), CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G.T.Hermanson etc., (1993), IMMOBILIZED AFFINITYLIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, R.L. etc., Eds.POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol.469, American Chemical Society, Washington, D.C.1991)
Many water-soluble polymers are well known by persons skilled in the art and can be used in the practice of the present invention.The term water-soluble polymer comprises as sugar (for example glucosan, amylose, hyaluronic acid, poly-(sialic acid), heparan, heparin or the like); Poly-(aminoacid); Nucleic acid; Synthetic polymer (for example gathers (acrylic acid), gathers (ether), for example gathers (ethylene glycol); The kind of peptide, albumen or the like.The present invention can use water-soluble polymer practice arbitrarily, and unique restriction is that polymer must comprise the point that remaining conjugate can connect.
The method of activated polymer can also be at WO 94/17039, U.S. the patent No. 5,324,844, WO 94/18247, WO 94/04193, U.S. the patent No. 5,219,564, U.S. the patent No. 5,122,614, WO 90/13540, U.S. the patent No. 5,281,698 and WO 93/15189 in find and the combination between activatory polymer and the peptide, blood coagulation factor VIII (WO 94/15625) for example, hemoglobin (WO 94/09027), oxygen carrier molecule (the U.S. patent No. 4,412,989), ribonuclease and superoxide dismutase (Veronese etc., App.Biochem.Biotech.11:141-45 (1985)).
Preferred water-soluble polymer is the polymer that most polymer molecule has approximately identical molecular weight in those polymer samples; Such polymer is called " the single dispersion ".The present invention by poly-(ethylene glycol) or single methoxy poly-(ethylene glycol) (m-PEG) conjugate further describe.The summary and the monograph that can obtain some PEG functionalization and put together.For example referring to, Harris, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); Wong etc., Enzyme Microb.Technol.14:866-874 (1992); Delgado etc., Critical Reviews inTherapeutic Drug Carrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995); And Bhadra etc., Pharmazie, 57:5-29 (2002).
Poly-(ethylene glycol) that can be used for forming conjugate of the present invention is linear or branched.
For example the water-soluble polymer of PEG, m-PEG, PPG and m-PPG also can improve the interior half-life of body or the area under curve (AUC) of therapeutic glycopeptide, for example, proteic PEG (PEGization) or m-PEG (m-PEGization) chemical modification can improve their molecular size and reduce their the surperficial accessibility and the accessibility of functional group, and this all depends on the size of the PEG that is connected with albumen.This has caused the plasma half-life or AUC and the Proteolytic enzyme stability that improve, and causes reduction (Chaffee etc., the J.Clin.Invest.89:1643-1651 (1992) of immunogenicity and liver picked-up; Pyatak etc., Res.Commun.Chem.PatholPharmacol.29:113-127 (1980)).Reported that the PEGization of interleukin-2 has improved its anti-tumor in vivo potential (Katre etc., Proc.Natl.Acad.Sci.USA.84:1487-1491 (1987)) and F (ab ') 2 the PEGization that derives from monoclonal antibody A7 improved its tumor-localizing (Kitamura etc., Biochem.Biophys.Res.Commun.28:1387-1394 (1990)), therefore, in another preferred embodiment, increase with respect to half-life or AUC in the body of deutero-peptide not by the half-life in the body of the deutero-peptide of method of the present invention with water-soluble polymer.
The raising of half-life or AUC preferably is expressed as the percentage range of the increase of this amount in the body of peptide.The percentage ratio that increases following is limited to about 40%, about 60%, about 80%, about 100%, about 150% or about 200%, is limited to about 60%, about 80%, about 100%, about 150% or be higher than about 250% on the scope.
The peg moiety of any molecular weight, for example 5kD, 10kD, 20kD and 30kD all can be used for method of the present invention.
Biomolecule
In another preferred embodiment, modify sugar and have biomolecule.In further preferred implementation, this biomolecule is functional protein, enzyme, antigen, antibody, peptide, nucleic acid (being mononucleotide or nucleoside, oligonucleotide, polynucleotide and strand or multichain nucleic acid), agglutinin, receptor or its combination.
Preferred biomolecule is not fluorescent basically, or emission seldom fluorescence and be unsuitable for the fluorescent labeling in performing an analysis.And the usually preferred biomolecule that adopts non-sugar, this preferred exception be adopt by with the covalently bound naturally occurring sugar of modifying of another entity (for example PEG, biomolecule, therapeutic part, diagnosis part etc.).In an illustrative embodiments, be connected with the connexon arm as the sugar moieties of biomolecule, sugar-connexon arm box is connected via method of the present invention with peptide subsequently.
The biomolecule that can be used for practice of the present invention can be from any source.Biomolecule can be separated maybe from natural origin and can produce by synthetic method, and peptide can be native peptides or mutant peptide.Sudden change can realize with chemomorphosis effect, direct mutagenesis or other induced mutation mode well known by persons skilled in the art.The peptide that is used for practice of the present invention comprises for example enzyme, antigen, antibody and receptor.Antibody can be polyclone or monoclonal; Complete or fragment.Peptide is the product of orthogenesis program alternatively.
Naturally occurring and synthetic peptide and nucleic acid all can be used for the present invention; These molecules can be connected with saccharide residue component or cross-linking agent by any available active group.For example, peptide can pass through reactive amine, carboxyl, sulfydryl or hydroxyl connection.It is terminal or in the intrachain site of peptide that active group can be positioned at peptide.Nucleic acid can pass through active group on the base (for example ring outer amine (exocyclicamine)) or the available hydroxyl on the sugar moieties (for example 3 '-or 5 '-hydroxyl) connection.Peptide and nucleic acid chains can further be derived in one or more sites, and suitable active group is connected on the chain.Referring to Chrisey etc., Nucleic Acids Res.24:3031-3039 (1996).
In further preferred version, select biomolecule to make peptide point to particular organization, thereby improve the conveying of peptide in organizing with respect to the quantity of the underivatized peptide that flows to this tissue by method modification of the present invention.In another preferred scheme, the derivatization peptide in seclected time, be fed to particular organization quantity since derivatization improve at least about 20%, be more preferably approximately at least 40%, be more preferably about at least 100%.At present, the preferred biomolecule of targeting application comprises the aglucon of antibody, hormone and cell surface receptor.
In the exemplary specific embodiment in addition, provide conjugate with biotin.Therefore, the avidin or the optionally biotinylated peptide of streptavidin part elaborate that have one or more modification groups by connection.
The therapeutic part
In another preferred scheme, modify steamed bun stuffed with sugar and draw together the therapeutic part.It will be appreciated by those skilled in the art that between the kind of therapeutic part and biomolecule, have overlapping; Many biomolecule have therapeutic properties or potential.
Therapeutic part can be the reagent of the clinical use accepted, and perhaps they can be the medicines of tentative use, or are studying the medicine of its activity or the mechanism of action.The therapeutic part can have the effect of checking in given morbid state, perhaps can only infer the effect that needs that demonstrates in given morbid state.In a preferred scheme, therapeutic partly is a chemical compound, the interactional ability of that screening them and selected tissue.The therapeutic of using in practice of the present invention partly comprises the medicine from the medicament categories that has multiple pharmacological activity in a large number.Preferred therapeutic part is not fluorescent basically, or emission in a small amount fluorescence and be unsuitable for the fluorescent labeling in performing an analysis.And usually preferred employing is not the therapeutic part of sugar.A kind of this preferred exceptional case be utilize by with another entity, for example covalently bound and sugar modified of PEG, biomolecule, therapeutic part, diagnosis part or the like.In another exemplary specific embodiment, the therapeutic sugar moieties is connected with the connexon arm, and sugar-connexon arm box is connected via method of the present invention with peptide subsequently.
Well-known for those skilled in the art therapeutic and diagnostic reagent with the method that various other kinds are connected, for example referring to, Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; And Dunn etc., Eds.POLYMERICDRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C.1991.
In an exemplary specific embodiment, the therapeutic part is connected via cracked key under selected state with modification sugar.Exemplary condition exists (for example esterase, reductase, oxidase), light, heat or the like including, but not limited to the pH that selects (for example in stomach, the small intestinal, cell intracellular vesicle), activating enzymes.The group of many cleavables known in the art, for example referring to, Jung etc., Biochem.Biophys.Acta, 761:152-162 (1983); Joshi etc., J.Biol.Chem., 265:14518-14525 (1990); Zarling etc., J.Immunol., 124:913-920 (1980); Bouizar etc., Eur.J.Biochem., 155:141-147 (1986), Park etc., J.Biol.Chem., 261:205-210 (1986); Browning etc., J.Immunol., 143:1859-1867 (1989).
The kind of available therapeutic part comprises for example anti-inflammatory drug of on-steroidal (NSAIDS).NSAIDS can select from for example following kind apoplexy due to endogenous wind: (for example propanoic derivatives, acetogenin, fragrant that acid (fenamic acid) derivant, biphenylcarboxylic acid derivatives and oxicam); The steroid anti-inflammatory drug that comprises hydrocortisone or the like; Antihistamine drug (for example chlorpheniramine, triprolidine); Antitussive (for example dextromethorphan, codeine, Ethanedisulfonate and pentoxyverine); Antipruritic agent (for example methdilazine and alimemazine); The medicine of anti-parasympathetic nervous physiological action (for example scopolamine, atropine, melyltropeine, levodopa); Bendectin and antinauseant (for example cyclizine, meclizine, chlorpromazine, buclizine); Anorexigenic (benzfetamine, phentermine, chlorphentermine, fenfluramine); Central stimulants (for example amphetamine, dexoxyn, dexamfetamine and methylphenidate); Anti-arrhythmic (for example Propranolol, procainamide, disopyramide, quinidine, encainide); Beta-adrenergic blocking agent (for example metoprolol, acebutolol, betaxolol, labetalol and timolol); Anti-heart failure medicine (for example milrinone, amrinone and dobutamine); Antihypertensive (for example enalapril, clonidine, hydralazine, minoxidil, guanadrel, guanethidine); Diuretic (for example amiloride and hydrochlorothiazide); Vasodilation (for example diltiazem , amiodarone, isoxsuprine, buphenine, tolazoline and verapamil); Vasoconstrictor (for example dihydroergotamine, Ergotamine and methysergide); Anti-ulcer medicament (for example ranitidine and cimetidine); Anesthetis (for example lignocaine, bupivacaine, chloroprocaine, cincaine); Antidepressant drug (for example imipramine, desipramine, amitriptyline, nortryptiline); Tranquilizer and tranquilizer (for example dissolve in chlorine nitrogen , benactyzine, benzquinamide, chlorine west, hydroxyzine, loxapine and promazine); Psychosis (for example chlorprothixene, fluphenazine, haloperidol, molindone, thioridazine and trifluoperazine); Antimicrobial drug (antibacterium, antifungal, protozoacide and antiviral drugs).
The antimicrobial drug of preferably incorporating this compositions into comprises, for example the beta-lactam medicine, quinolone medicine, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, many third rings are plain, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, the different thiosulfuric acid of hexamidine, metronidazole, pentamidine, gentamycin, kanamycin, lineomycin, metacycline, hexamethylenamine, minocycline, neomycin, netilmicin, paromomycin, streptomycin, tobramycin, the pharmaceutically acceptable salt of miconazole and amantadine.
Other can be used for drug moiety in the practice of the present invention and comprises antineoplastic agent (for example androgen antagonist (for example leuproside or flutamide), cytocide (for example 14-Hydroxydaunomycin, doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, β-2-interferon) estrogen antagonist (for example tamoxifen), antimetabolite (for example fluorouracil, methotrexate, mercaptopurine, thioguanine).Be also included within these classifications be diagnosis and treatment based on radioisotopic reagent, and the toxin of puting together, for example ricin, geldanamycin, mytansin, CC-1065, duocarmycins, Chlicheamycin and its dependency structure and analog.
The therapeutic part can also be hormone (for example medroxyprogesterone, estradiol, leuproside, megestrol, octreotide or a somatostatin); Muscle relaxant (for example cinnamedrine, cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine, idaverine, ritodrine, diphenoxylate, dantrolene and azumolene); Spasmolytic; Bone-activatory medicine (for example diphosphate and phosphine acyl-alkyl phosphinic acid medicaments compound); Endocrine regulation medicine (for example contraceptive (for example etynodiol, ethinylestradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone), diabetes regulator (for example glibenclamide or chlorpropamide), anabolism medicine, for example testolactone or stanozolol, androgen (for example methyltestosterone, testosterone or fluoxymesterone), antidiuretic (for example Desmopressin) and calcitonin.
Also can adopt estrogen (for example diethylstilbestrol), glucocorticoid (for example triamcinolone, betamethasone or the like) and progestogens among the present invention, as norethindrone, etynodiol, norethindrone, levonorgestrel; Thyroid (for example liothyronine or levothyroxine) or antithyroid drug (for example methimazole); Anti-high lactotropin liberin medicine (for example cabergoline); Hormone inhibitors (for example danazol or goserelin), odinagogue (for example methylergometrine or oxytocin) and prostaglandin, for example mioprostol, Alprostadil or dinoprostone also can adopt.
Other adoptable modification group comprise immunomodulator (for example hydryllin, mast cell stabilizers, as lodoxamide and/or cromolyn, steroid (for example triamcinolone, beclometasone, cortisone, dexamethasone, prednisolone, methylprednisolone, beclometasone, or clobetasol), histamine h2 antagonist (for example famotidine, cimetidine, ranitidine), immunosuppressant (for example azathioprine, cyclosporin) or the like.Also can adopt group such as sulindac, etodolac, ketoprofen and ketorolac with anti-inflammatory activity.The medicine that other and the present invention together use it will be apparent to those skilled in the art that.
Modify the preparation of sugar
Usually, sugar moieties and modification group link together by using active group, and this active group generally changes into nonreactive new organic functions group or kind under the physiology corresponding condition by method of attachment.Sugar reactive functional group cumularsharolith is on any site of sugar moieties.Being used for the active group of practice of the present invention and reaction classification, to put together chemical field at biology usually be well-known.The reaction classification of at present available preferred reactive sugars part is to carry out under gentle relatively condition.These are including, but not limited to nucleophilic displacement of fluorine (for example amine and alcohol and acyl halide, Acibenzolar reaction), electrophilic substitution (for example enamine reaction) and add carbon-to-carbon and carbon-hetero atom multiple bond (for example Michael addition reaction, Di Ersi-A Erde reaction).These and other available side should discuss in following document, for example, and March, ADVANCEDORGANIC CHEMISTRY, 3rd Ed., John Wiley; Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; With Feeney etc., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol.198, American Chemical Society, Washington, D.C., 1982.
Include but not limited to from sugar nuclear or the outward extending effective reactive functional group of modification group:
(a) the carboxyl derivant different with it includes but not limited to, N-hydroxy-succinamide ester, N-hydroxybenzotriazole ester, acid halide, acylimidazole, thioesters, p-nitrophenyl ester, alkyl, alkenyl, alkynyl and aromatic ester;
(b) hydroxyl, it can become, for example ester, ether, aldehyde or the like.
(c) halogenated alkyl group, wherein halogenide can replace with nucleophilic group such as amine, carboxylate anion, mercaptan anion, carbanion or alkoxide ion afterwards, thereby caused new group covalently bound on the functional group of halogen atom;
(d) dienophile group can participate in Diels-Alder reaction, for example, and the dimaleoyl imino group;
(e) aldehydes or ketones base, therefore deriving and may realize subsequently via the formation of carbonyl derivative, for example, imines, hydrazone, semicarbazones or oxime, or via the mechanism as Ge Liya addition or lithium alkylide addition;
(f) sulfuryl halide group carries out subsequently the reaction with amine, for example, forms sulfonamides;
(g) mercapto can for example become disulphide or reacts with acyl halide;
(h) amine or sulfydryl for example can be acidylate, alkylation or oxidation;
(i) alkene for example can carry out cycloaddition, acylation, Michael addition or the like; And
(J) epoxide can react with for example amine and hydroxy compounds.
Can selective response sexual function group they are not participated in or do not interfere assembling reactive sugars nuclear or the necessary reaction of modification group.In addition, can make by the existence of protecting group reactive functional group protected and do not participate in the reaction.Those skilled in the art understand how to protect specific functional group, and it is not conflicted with the reaction condition of selecting.The effective example of protecting group, for example referring to Greene etc., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, JohnWiley ﹠amp; Sons, New York, 1991.
In discussion subsequently, be used for the example of the specific modification sugar of practice of the present invention with setting forth some.In the exemplary specific embodiment, use sialic acid derivative to examine as sugar, modification group is attached thereto.Be absorbed in that sialic acid derivative is discussed only is clear for what illustrate, and should not be construed as limitation of the scope of the invention.It will be appreciated by those skilled in the art that similar mode that multiple other sugar moieties also can use sialic acid to set forth for example activates and derives.For example, can obtain the method for many modification galactose, glucose, N-acetylgalactosamine and fucose, only for the example of several sugared substrates, the method that can easily be generally acknowledged by prior art is modified.For example referring to, Curr.Med.Chem.6:93 such as Elhalabi (1999); With Schafer etc., J.Org.Chem.65:24 (2000)).
In a kind of exemplary specific embodiment, the peptide of being modified by method of the present invention is at prokaryotic cell (for example escherichia coli), eukaryotic cell, comprise yeast and mammalian cell (for example Chinese hamster ovary celI) or the glycopeptide that in genetically modified animal, produces, therefore contain sialylated by halves N-and/or O-and connect oligonucleotide chain.Lack sialic acid and contain the oligonucleotide chain of the glycopeptide of terminal galactose residues, can be by PEGization, PPGization or with the sialic acid change of modification.
Exemplary PEG-sialic acid comprises:
Wherein L is an assorted alkyl connexon part alkyl replacement or unsubstituted or replacement or unsubstituted, and its sialic acid part and peg moiety couple together, and " n " is 1 or bigger numeral; And
Sign " s " representative from 0 to 20 integer wherein, " n " are 1 or bigger numeral.
In scheme 4, handle aminoglycoside 1 with the Acibenzolar of aminoacid (for example glycine) derivant of protecting, the osamine residue is changed into the amino acid amide adduct of corresponding protection.Handle adduct with aldolase and form alpha-hydroxy carboxylic acid compounds 2, under the effect of CMP-SA synzyme, chemical compound 2 becomes corresponding C MP derivant, produces chemical compound 3 by catalytic hydrogenation CMP derivant then.The amine that employing is introduced via the formation of glycine adduct carries out the site that PEG is connected as reacting with chemical compound 3 with activatory PEG or PPG derivant (for example PEG-C (O) NHS, PEG-OC (O) O-p-nitre phenyl), produces the kind as 4 or 5 respectively.
Scheme 4
Table 1 has been listed the representative instance with PEG or the single phosphoric acid of the deutero-sugar of PPG part.Human growth hormone's mutant can be modified by the method for scheme 1, the method preparation that other derivant is generally acknowledged by prior art.For example referring to, Keppler etc., Glycobiology 11:11R (2001); And Charter etc., Glycobiology 10:1049 (2000).Reactive PEG of other amine and PPG analog can be bought from the market and obtain, and maybe can prepare by the method that those skilled in the art are easy to get.
The sugared phosphoric acid that is used for the modification of practice of the present invention can be substituted in other site and above-mentioned site.At present preferred sialic being substituted among the formula I illustrated:
Wherein X is a linking group, be preferably selected from-O-,-N (H)-,-S, CH2-and-N (R)
2, wherein each R is independently selected from R
1-R
5The member, each representative of symbol Y, Z, A and B is selected from the above-mentioned group identical with X.Select X, Y, Z, A and B independently of one another, so they can be identical or different.Symbol R
1, R
2, R
3, R
4And R
5Represent H, water-soluble polymer, therapeutic part, biomolecule or other parts.In addition, these symbologies and water-soluble polymer, therapeutic part, biomolecule or the bonded connexon of other parts.
The exemplary part that is connected with conjugate disclosed herein includes but not limited to PEG derivant (alkyl-PEG, acyl group-PEG, acyl group-alkyl-PEG, alkyl-acyl group-PEG, carbamyl-PEG, aromatic radical-PEG), PPG derivant (for example alkyl-PPG, acyl group-PPG, acyl group-alkyl-PPG, alkyl-acyl group-PPG carbamyl-PPG, aryl-PPG), therapeutic part, diagnosis part, Man-6-P, heparin, heparan, SLe for example
x, mannose, Man-6-P, saliva acidic group Lewis X, FGF, VFGF, albumen, chrondroitin, Keratin, dermatan, albumin, integration element, antenniferous oligosaccharide, peptide or the like.The method of attachment of various modification groups and sugar moieties is (POLY (the ETHYLENE GLYCOL CHEMISTRY:BIOTECHNICAL AND BIOMEDICALAPPLICATIONS that are easy to get to those skilled in the art, J.Milton Harris, Ed., Plenum Pub.Corp., 1992; POLY (ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J.Milton Harris, Ed., ACS Symposium Series No.680, AmericanChemical Society, 1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; And Dunn etc., Eds.POLYMERICDRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C.1991).
Crosslinked group
The preparation that is used for the modification sugar of method of the present invention comprises and modification group is connected with saccharide residue and forms stable adduct that this adduct is the substrate of glycosyl transferase.Sugar can be connected by zero level or senior cross-linking agent with modification group.The exemplary bifunctional chemical compound that can be used to connect modification group and carbohydrate part includes but not limited to bifunctional poly-(ethylene glycol), polyamide, polyethers, polyester or the like.The method in common that connects carbohydrate and other molecule is known in the literature.For example referring to, Lee etc., Biochemistry 28:1856 (1989); Bhatia etc., Anal.Biochem.178:408 (1989); Janda etc., J.Am.Chem.Soc.112:8886 (1990) and Bednarski etc., WO92/18135.In discussion subsequently, carry out gentleness on the sugar moieties of active group modification sugar in the early stage and handle.The focus of this discussion is clear for what illustrate, it will be appreciated by those skilled in the art that this discussion is also relevant with the active group on the modification group.
A kind of exemplary strategy relates to and uses isodigeranyl functional cross-link agent SPDP (n-succinimido-3-(2-pyridine radicals dithio) propionic ester) that the sulfydryl of protection is added on the sugar, make then sulfydryl go to protect with modification group on another sulfydryl form disulfide bond.
If SPDP influences the ability of sugar as the glycosyl transferase substrate of modifying unfriendly, in also available other a series of cross-linking agent one forms disulfide bond as 2-iminothiolane or N-butanimide S-acetyl thio acetic acid (SATA).2-iminothiolane and primary amine reaction are containing the not shielded sulfydryl of integration on the molecule of amine immediately.SATA also with primary amine reaction, but integrate the sulfydryl of protection, use the azanol deacetylation later on, produce free sulfhydryl group.In all cases, the sulfydryl of integration freely with the reaction of the sulfydryl of other sulfydryl or protection, as SPDP, form the disulfide bond that needs.
Above-mentioned strategy is exemplary, but the connexon that is not limited to adopt among the present invention.Also can obtain to be used to make other cross-linking agent of the crosslinked different strategy of modification group and peptide.For example; (((S-(2-sulfo-pyridine) sulfydryl-propiono hydrazides) and the carbohydrate partial reaction of being handled oxidation in advance by the periodic acid of gentleness, therefore the hydrazides at cross-linking agent partly and between the acetaldehyde of periodic acid generation forms the hydrazone key to TPCH for S-(2-sulfo-pyridine)-L-cysteine hydrazides and TPMPH.TPCH and TPMPH introduce the sulfydryl of 2-pyridine radicals thioketone protection on sugar, it can go protection with DTT, is used for subsequently puting together, as form disulfide bond between component.
If find that disulfide bond is unsuitable for producing stable modification sugar, can adopt other cross-linking agent between component, to form more stable key.Thereby on component, introduce maleimide base group isodigeranyl functional cross-link agent GMBS (N-γ-maleoyl imido butyryl acyloxy) butanimide) and SMCC (succinimido 4-(N-maleoyl imido-methyl) cyclohexane extraction) and primary amine reaction.Maleimide base group can be subsequently with another component on the sulfydryl reaction, described sulfydryl can import by previously mentioned cross-linking agent, forms stable thioether bond thus between component.As the steric influence between the fruit component component active or modify the ability of sugar as the glycosyl transferase substrate, can use the cross-linking agent of between component, introducing long spacer arm, comprise the derivant (being SPDP) of some above-mentioned cross-linking agent.Therefore enough suitable available cross-linking agent are arranged, and wherein the selection of each depends on that all it is to the peptide conjugate of optimum with modify the effect that sugar produces and select.
Many reagent can be used for modifying modification saccharic composition with intramolecular chemical crosslinking, and (summary of cross-linking reagent and cross-linking process is referring to Wold, F., Meth.Enzymol.25:623-651,1972; Weetall, H.H., and Cooney, D.A., In:ENZYMESAS DRUGS. (Holcenberg, and Roberts, eds.) pp.395-442, Wiley, NewYork, 1981; Ji, T.H., Meth.Enzymol.91:580-609,1983; Mattson etc., Mol.Biol.Rep.17:167-183,1993, all documents are all introduced as reference herein).Preferred cross-linking agents stems from various zero-length, with cross-linking agent bifunctional and the isodigeranyl function.Zero-length cross-linking agent comprises two direct combinations of intrinsic chemical group, and does not import external material.The reagent that the catalysis disulfide bond forms belongs to this kind.Another example is the reagent of inducing carboxyl and primary amino radical formation amido link, as carbodiimide, ethyl chloroformate, Woodward ' s reagent K (the 2-ethyl-different azoles of 5-phenyl -3 '-sulfonic acid) and N,N'-carbonyldiimidazole.Except that these chemical reagent, T-5398 (glutamyl-peptide-gamma glutamyltransferase; EC 2.3.2.13) can be used as distance of zero mark degree cross-linking agent.These enzymes are the catalyzing acyl transfer reaction in the acylamino-group of protein binding glutamine residue, uses primary amino radical as substrate usually.Preferred homotype contains two identical or two different sites respectively with special-shaped bifunctional reagent, its can with amino, sulfydryl, guanidine radicals, indole or nonspecific radical reaction.
The amino-reactive group
In a preferred scheme, the site on the cross-linking agent is the amino-reactive group.The non-limiting instance of available amino-reactive group comprises carbonate ester, N-hydroxy-succinamide (NHS) ester, imino-ester, isocyanates, acyl halide, aromatic radical azide, p-nitrophenyl ester, aldehyde and sulfonic acid chloride.
NHS ester and the amino preferential reaction primary (comprising aromatic series) of modifying saccharic composition.Known and the primary amine competitive reaction of the imidazole radicals of histidine, but hydrolysis takes place in product instability easily.This reaction relates to the nucleophillic attack of amine to the acid carboxyl of NHS ester, forms amide, discharges N-hydroxy-succinamide.Thereby, lost the positive charge of original amino.
Imino-ester is the most specific acylating reagent that reacts with the amido of modifying saccharic composition.Between pH7 and 10, imino-ester only with primary amine reaction.Imidic acid is attacked on primary amine nucleophilicity ground, being created in high pH is decomposed into the intermedium of amidine or is hanging down new imidic acid under the pH, new imidic acid can with another primary amine reaction, thereby crosslinked two amine groups, a kind of infer have difunctional reactive single function imidic acid.With the primary product of primary amine reaction be the stronger amidine of amine alkalescence compared with the beginning, thereby kept the positive charge of initial amino.
Isocyanates (and isothiocyanate) and the primary amine reaction of modifying saccharic composition form stable key.They produce relative unsettled product with the reaction of sulfydryl, imidazoles and tyrosyl group.
Also as amino specific reagent, wherein the nucleophilic amine of affinity component is attacked acid carboxyl to acyl azide under the alkalescence situation, for example at pH8.5.
For example 1,5-two fluoro-2, the aromatic radical halogenide of 4-dinitro benzene preferential with amine groups of modifying saccharic composition and the reaction of tyrosine phenylol, also with sulfydryl and imidazole radicals reaction.
P-nitrophenyl ester single and dicarboxylic acids also is effective amino-reactive group.Although the reagent specificity is not really high, α-seem that with epsilon-amino group reaction is the rapidest.
Aldehyde and the primary amine reaction of modifying sugar as glutaraldehyde.Although the reaction of the aldehyde radical of amine groups and aldehyde forms unsettled Schiff's base, glutaraldehyde can be modified sugar by enough stable crosslinked changes.At pH6-8, under the promptly typical crosslinked pH condition, the cyclic polymer experience is dewatered and is formed the unsaturated aldehyde polymer of alpha-beta.Yet when being connected with another pair key, Schiff's base is stable.The Resonant Interaction of two two keys has prevented the hydrolysis of Schiff key, and the amine of high local concentrations can be attacked the two keys of alkene and form stable Michael addition compound product.
Aromatic sulfonyl and many sites reaction of modifying saccharic composition, but with the reaction of amine groups be most important, the result forms stable sulfonamides key.
The sulfydryl reactive group
In another preferred scheme, the site is the sulfydryl reactive group.Effectively, the example of nonrestrictive sulfydryl reactive group comprises maleimide, alkyl halide, pyridyl disulfide and sulfo-phthalimide.
Maleimide and the sulfydryl preferential reaction of modifying saccharic composition form stable thioether bond.They also react with the imidazole radicals of slack-off greatly speed and primary amino radical group and histidine.Yet at pH7, maleimide base group can be thought sulfur hydrogen specificity group, because under this pH, the reaction rate of simple mercaptan is bigger 1000 times than the reaction rate of corresponding amine.
Alkyl halide and sulfydryl, sulfide, imidazoles and amine groups reaction.Yet, arrive under the alkalescence pH the main and stable thioether bond of sulfydryl reaction formation of alkyl halide in neutrality.At higher pH, tend to reaction with amine groups.
Pyridyl disulfide produces blended disulphide via disulfide exchange and free sulfhydryl group reaction.As a result, pyridyl disulfide is the most specific sulfydryl reactive group.
Sulfo-phthalimide and the reaction of free sulfydryl form disulphide.
The carboxyl-reactive residue
In another embodiment, water-soluble and carbodiimide organic solvent is used as carboxyl-reactive reagent.The reaction of these chemical compounds and free carboxyl group forms false carbamide, false carbamide can with available amine coupling, produce amido link, how instruction modifies carboxyl (Yamada etc., Biochemistry 20:4836-4842,1981) with carbodiimide.
Except that utilizing site-specific reactive moieties, the present invention's design couples together sugar and modification group by utilizing non-specific active group.Exemplary non-specific cross-linking agent comprises the photoactivation group, and it does not have activity in the dark fully, becomes reactive species behind the photon that absorbs appropriate energy.In a preferred scheme, the photoactivation group is selected from the precursor of the nitrene that produces behind heating or photodissociation azide.No electronics nitrene is very active, can comprise N-H, O-H, C-H and C=C reaction with many chemical bonds.Although can adopt the azide (aryl, alkyl and acyl derivative) of three types, aromatic radical azide most preferably at present.The reactivity of the aromatic radical azide under photolysis is stronger to N-H and O-H comparison c h bond.The arylnitrenes of no electronics promptly widening of the ring forms dehydrogenation azepine , trend and nucleopilic reagent reaction, rather than formation C-H inserts product.The reactivity of aromatic radical azide can be improved as the nitro in the ring or the existence of hydroxyl by electron-withdrawing substituent, and such substituent group promotes the longer wavelength of absorption maximum deflection of aromatic radical azide.Unsubstituted aromatic radical azide has the absorption maximum in the 260-280nm scope, and hydroxyl and nitryl aromatic base azide significantly absorb the light that surpasses 305nm.Therefore, most preferably hydroxyl and nitryl aromatic base azide are because they adopt condition than the photolysis of the less injury of unsubstituted aromatic radical azide to the affinity component.
In another preferred scheme, the photoactivation group is selected from fluorizated aromatic radical azide.The photolysis product of fluoridizing the aromatic radical azide is the inferior trip base of armaticity amine (nitrene), the all inferior trip of these armaticity amine bases all experience the high efficiency characteristic reaction of these groups, comprise that c h bond inserts (Keana etc., J.Org.Chem.55:3640-3647,1990).
In another embodiment, the photoactivation group is selected from the benzophenone residue.Benzophenone reagent produces higher crosslinked output than aromatic radical azide reagent usually.
In another embodiment, the photoactivation group is selected from diazonium compound, and it forms no electronics Cabbeen under photolysis.These Cabbeens carry out many reactions, comprise inserting c h bond, be added into two keys (comprising the aromatic series system), attract hydrogen and produce carbon ion with the nucleophilic center coordination.
In another embodiment, the photoactivation group is selected from the diazonium acetone acid, and for example, the p-nitrophenyl ester of p-nitrophenyl diazonium acetone acid and fatty amine reaction produce diazonium acetone acid amide, form aldehyde after ultraviolet photodissociation effect.The affinity component that the diazonium acetone acid of photolysis is modified will as formaldehyde or glutaraldehyde react form crosslinked.
Homotype bi-functional cross-linking agent with primary amine reaction
Synthetic, the character of amine-reactant cross-linker and use the ground of commercialization in the literature and describe (to the summary of cross-linking process and reagent, referring to above).Can obtain many reagent (PierceChemical Company for example, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).
Preferred non-limiting instance with difunctional NHS ester comprises two butanimide 1,3-propanedicarboxylic acids (DSG), suberic acid two butanimides (DSS), two (sulfosuccinimide) suberic acid (BS), tartaric acid two butanimides (DST), tartaric acid disulfo butanimide (sulfo group-DST), two [2-(succinimido oxygen ketonic oxygen)-ethyl]-sulfones (BSOCOES), two-2-(sulfosuccinimide oxygen base ketonic oxygen base) ethyl sulfone (sulfo group BSOCOES), ethylene glycol bisthioglycolate (succinimido succinate) (EGS), ethylene glycol bisthioglycolate (sulfosuccinimide base succinate) (sulfo group EGS), dithio two (succinyl phosphorons amino propyl acid ester) (DSP) and dithio two (sulfosuccinimide propanoic acid) (sulfo group DSP).Preferred nonrestrictive example with difunctional imino-ester comprises dimethyl maleimide acid esters (DMM), dimethyl butanimide acid esters (DMSC), dimethyl adipimide acid esters (DMA), dimethyl-g imidodicarbonic diamide acid esters (DMP), dimethyl-octa imidodicarbonic diamide acid esters (DMS), dimethyl-3,3 '-oxygen dipropyl acid imide acid esters (DODP), dimethyl-3,3 '-(methylene dioxygen base) dipropyl acid imide acid esters (DMDP), dimethyl-3 '-(dimethylene dioxygen base) dipropyl acid imide acid esters (DDDP), dimethyl-3,3 '-(tetramethylene dioxygen base) dipropyl acid imide acid esters (DTDP), and dimethyl-3,3 '-the two propionyl imidoethers (DTBP) of dithio.
The example of the difunctional isothiocyanate of preferred nonrestrictive homotype comprises: bitoscanate (DITC) and 4,4 '-two isothiocyanos-2,2 '-disulfonic acid Stilbene (DIDS).
The example of the difunctional isocyanates of preferred nonrestrictive homotype comprises dimethylbenzene-vulcabond, Toluene-2,4-diisocyanate, 4-vulcabond, Toluene-2,4-diisocyanate-isocyanates-4-isothiocyanate, 3-methoxyl group diphenyl methane-4,4 '-vulcabond, 2,2 '-two carboxyls-4,4 '-phenylazo vulcabond and hexamethylene diisocyanate.
The example of the difunctional aryl halide of preferred nonrestrictive homotype comprises 1,5-two fluoro-2, and 4-dinitro benzene (DFDNB) and 4,4 '-two fluoro-3,3 '-dinitrophenyl-sulfone.
The example of the difunctional fat-based aldehyde reagent of preferred nonrestrictive homotype comprises Biformyl, malonaldehyde and glutaraldehyde.
The example of the difunctional acylating reagent of preferred nonrestrictive homotype comprises the nitro phenyl ester of dicarboxylic acids.
The example of the difunctional aromatic sulfonyl of preferred nonrestrictive homotype comprises phenol-2,4-disulfonic acid chloride, and alpha-Naphthol-2, the 4-disulfonic acid chloride.
The example of preferred nonrestrictive other amino-reactive homotype bifunctional reagent comprises and the amine reaction produces the two carbonic acid of two carbamic erythritols.
Homotype bi-functional cross-linking agent with free sulfydryl reaction
(summary of cross-linking process and reagent, referring to above) described in synthetic, the character and the application of such reagent in the literature.(PierceChemical Company for example, Rockford, Ill. can be bought and obtained to many reagent on market; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).
The example of the difunctional maleimide of preferred nonrestrictive homotype comprises BMI hexane (BMH), N, N '-(1,3-penylene) BMI, N, N '-(1,2-penylene) BMI, phenylazo BMI and two (N-maleimide methyl) ether.
The example of the difunctional pyridine disulphide of preferred nonrestrictive homotype comprises 1,4-is two-3 '-(2 '-the two sulfur of pyridine radicals) propionamido butane (DPDPB).
The example of the difunctional alkyl halide of preferred nonrestrictive homotype comprises 2,2 '-two carboxyls-4,4 '-two iodacetyl ammonia diphenyl diimides, α, α '-two iodo-xylol sulfonic acid, α, α '-two bromo-xylol sulfonic acid, N, N '-two (b-bromoethyl) benzyl amine, N, N '-two (acetyl bromide) phenylhydrazine and 1, two (acetyl bromide) the amino 3-phenylpropyl alcohol alkane of 2-.
The difunctional photoactivated cross-linking agent of homotype
(summary of cross-linking process and reagent referring to above) described in synthetic, the character and the application of such reagent in the literature.Some reagent are commercially available (for example, PierceChemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).
The example of the difunctional photoactivated cross-linking agent of preferred nonrestrictive homotype comprises two-β-(4-azidosalicylic acid acyl ammonia) ethyl disulphide (BASED), two-N-(2-nitro-4-phenylazide)-cystamine-S, S-dioxide (DNCO), with 4,4 '-the two aziminobenzenes of two sulfur.
The special-shaped bifunctional reagent of amino-reactive with pyridyl disulfide moieties
(summary of cross-linking process and reagent referring to above) described in synthetic, the character and the application of such reagent in the literature.Many reagent can be bought on market and obtain (for example, PierceChemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, OR).
Preferred nonrestrictive example with heterobifunctional agent of pyridine disulfide moieties and amino-reactive NHS ester comprises N-butanimide-3-(2-pyridine two sulfur) propionic ester (SPDP), butanimide 6-3-(2-pyridine two sulfur) propionic acid amide. caproic acid (LC-SPDP), thiosuccimide 6-3-(2-pyridine two sulfur) propionic acid amide. caproic acids (sulfo-LCSPDP), 4-butanimide oxygen carbonyl-Alpha-Methyl-α-(2-pyridine two sulfur) toluene (SMPT) and thiosuccimide 6-Alpha-Methyl-α-(2-pyridine two sulfur) toluamide caproic acid (sulfo--LC-SMPT).
The special-shaped bifunctional reagent of amino-reactive with maleimide amine moiety
Synthetic, the character and the application of such reagent are described in the literature.Preferred nonrestrictive example with heterobifunctional agent of maleimide amine moiety and amino-reactive NHS ester comprises butanimide maleimide acetic acid (AMAS), butanimide 3-maleimide propanoic acid (BMPS), N-γ-maleimide butyryl oxygen succinimide ester (GMBS), N-γ-maleimide butyryl oxygen sulfur succinimide ester (sulfo-GMBS), butanimide 6-maleimide caproic acid (EMCS), butanimide 3-maleoyl benzoic acid (SMB), m-maleimide benzoyl-N-hydroxy-succinamide ester (MBS), m-maleimide benzoyl-N-hydroxy thiosuccinimide ester (sulfo-MBS), butanimide 4-(N-maleimide methyl)-cyclohexane extraction-1-carboxylic acid (SMCC), thiosuccimide 4-(N-maleimide methyl) cyclohexane extraction-1-carboxylic acid (sulfo-SMCC), butanimide 4-(to maleimide phenyl) butanoic acid (SMPB) and thiosuccimide 4-(to maleimide phenyl) butanoic acid (sulfo-SMPB).
The special-shaped bifunctional reagent of amino-reactive with alkyl halide part
Synthetic, the character and the application of such reagent are described in the literature.Preferred nonrestrictive example with heterobifunctional agent of alkyl halide part and amino-reactive NHS ester comprises N-butanimide-(4-iodoacetyl) amino benzoic acid (SIAB); thiosuccimide-(4-iodacetyl) amino benzoic Acid (sulfo-SIAB); butanimide-6-(iodacetyl) aminocaproic acid (SIAX); butanimide-6-(6-((iodoacetyl)-amino) hexanoyl ammonia) caproic acid (SIAXX); butanimide-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane extraction-1-carbonyl) aminocaproic acid (SIACX); and butanimide-4 ((iodoacetyl)-amino methyl cyclohexane extraction-1-carboxylic acid (SIAC).
The example that preferably has the heterobifunctional agent of amino-reactive NHS ester and alkyl dihalide part is a N-hydroxy-succinamide 2,3-dibromo-propionic acid (SDBP).SDBP introduces intramolecular crosslinked by the amine groups that connects it to the affinity component.Dibromo propionyl part is controlled (McKenzie etc., Protein Chem.7:581-592 (1988)) to the reaction of primary amine group by reaction temperature.
Preferred nonrestrictive example with special-shaped bifunctional reagent of alkyl halide part and amino-reactive p-nitrophenyl ester moiety comprises p-nitrophenyl iodine acetic acid (NPIA).
Known other the cross-linking agent of those skilled in the art, for example referring to, Pomato etc., the U.S. patent No. 5,965,106.The suitable crosslinking agent of selecting a kind of application-specific is in those skilled in the art's limit of power.
The connexon group of cleavable
In the specific embodiment in addition, by can cleavedly providing the connexon group with the group that discharges modification group from saccharide residue.The group of many cleavables known in the art, for example referring to, Jung etc., Biochem.Biophys.Acta 761:152-162 (1983); Joshi etc., J.Biol.Chem.265:14518-14525 (1990); Zarling etc., J.Immunol.124:913-920 (1980); Bouizar etc., Eur.J.Biochem.155:141-147 (1986); Park etc., J.Biol.Chem.261:205-210 (1986); Browning etc., J.Immunol.143:1859-1867 (1989).And bifunctional (homotype and special-shaped bifunctional) connexon group of a large amount of cleavables can be bought acquisition from the supplier as Pierce etc.
The part of exemplary cleavable can make and use up, heat or as the reagent cracking of mercaptan, azanol, alkali, periodic acid or the like.And, cleaved with endocytosis in vivo (for example, the cis-Aconitum carmichjaelii Debx. base of some preferred group; Referring to Shen etc., Biochem.Biophys.Res.Commun.102:1048 (1991)).Preferred cleavable group comprises the part of cleavable, is the member who is selected from the group that is made of disulphide, ester, imines, carbonic acid, nitrobenzyl, benzoyl group and benzoin group.
Modification sugar combines with peptide
Using suitable enzyme mediation to modify sugar is connected with glycosylated or nonglycosylated peptide.Preferably, select the concentration of donor sugar, enzyme and the receptor peptide of modification to make glycosylation proceed to exhaust up to receptor.Though hereinafter the item of Lun Shuing is illustrated under the situation of sialyltransferase, usually also applicable to other glycosyl transferase reaction.
The method of the oligosaccharide structure that some use glycosyl transferase synthetic need is known, and usually applicable to the present invention.For example WO 96/32491, Ito etc., and the Pure Appl.Chem.65:753 (1993) and the U.S. patent No. 5,352,670,5,374,541 and 5,545,553 have described exemplary method.
The present invention uses the combination of single glycosyl transferase or glycosyl transferase to carry out.For example, can use the combination of sialyltransferase and galactosyltransferase.In the specific embodiment of the enzyme that uses more than one, enzyme and substrate preferably make up in initial reaction mixture, or in case enzyme and the reagent that carries out second enzymatic reaction with regard to adding is finished or almost finished in first enzymatic reaction.By in a container, implementing two enzymatic reactions in turn, improved gross production rate than the method for separation of intermediates kind wherein.And, reduced the removing and the processing of extra solvent and side-product.
In a preferred version, first and second enzymes all are glycosyl transferases.In another preferred version, a kind of enzyme is an endoglycosidase.In other preferred version, adopt two or more enzymes to assemble the glycoprotein of modification of the present invention, enzyme is used for changing sugared structure in any site of peptide before or after adding modification sugar to peptide.
In another embodiment, this method is utilized one or more exoglycosidases or endoglycosidase.Usually glycosidase is a mutant, is transformed the formation glycosyl bond rather than with its cracking.The mutant dextranase generally includes the replacement of an amino acid residue to an active acidic amino acid residue site.For example, when the inscribe dextranase was endo-H, the active site residue of replacement is the Asp, Glu or its combination in site 132 in site 130 normally.Usually aminoacid replaces with serine, alanine, agedoite or glutamine.
The mutant enzyme catalytic reaction is undertaken by the synthesis step similar to the back reaction of inscribe dextranase hydrolysing step usually.In these specific embodiment, glycosyl donor molecule (for example, the widow or the monosaccharide structure that need) contains leaving group, and the GlcNAc residue of reaction on albumen adds donor molecule.For example, leaving group can be a halogen, as fluoride.In other specific embodiment, leaving group is Asn or Asn-peptide moiety.In the specific embodiment in addition, the GlcNAc residue on the glycosyl donor molecule is modified.For example, the GlcNAc residue can comprise 1,2 azoles quinoline part.
In a preferred version, each enzyme of the production conjugate of the present invention of use exists with catalytic amount.The catalytic amount of specific enzyme changes according to the concentration of the substrate of enzyme and reaction condition such as temperature, time and pH value.Determine that at the concentration of substrate of preliminary election and the catalytic amount of the given enzyme under the reaction condition be that those skilled in the art are well-known.
The temperature of carrying out above-mentioned steps can be from only above zero to the scope of the temperature of the most responsive enzyme denaturation.The scope of preferred temperature is approximately 0 ℃ to about 55 ℃, more preferably about 20 ℃ to about 30 ℃.In another exemplary specific embodiment, under the temperature that improves, use Zimadzhunt L 340 to implement one or more steps of this method.
Reactant mixture is kept one section is enough to make the glycosylated time of receptor, thereby forms the conjugate that needs.Some conjugates usually just can detect after several hours, just can obtain callable amount in common 24 hours or shorter time.Those skilled in the art understand reaction rate depends on some variable factors (for example enzyme concentration, donor concentration, acceptor density, temperature, solvent volume), and these factors are optimized a selected system.
The present invention also provides the production of the peptide of plant-scale modification.As use herein, commercial scale is produced at least approximately 250mg usually, preferably about at least 500mg, the final purification conjugate of more preferably about at least 1 gram, preferably after single reaction time, promptly conjugate is not from the combination of the product of same, continuous multiple synthesis cycle.
In discussion subsequently, the present invention illustrates with puting together of glycosylated peptide by modifying the sialic acid part.The sialic acid of exemplary modification m-PEG labelling.Sialic acid and glycosylated peptide that following main discussion PEG modifies are clear for what illustrate, and do not mean that hint the present invention is restricted to puting together of these two components.The technical staff understands this discussion usually applicable to the modification glycosyl part that adds except sialic acid.And discussion comprises other water-soluble polymer, therapeutic part and biomolecule similarly applicable to using other reagent except m-PEG to modify glycosyl unit.
Can use enzymatic method that the carbohydrate of m-PEGization or m-PPGization is optionally imported on peptide or the glycopeptide.This method adopts the modification sugar of the reactive functional group that contains PEG, PPG or shielding, and with suitable glycosyl transferase or the combination of sugared synthase.Glycosyl transferase by the carbohydrate key that select to produce needs and utilize and modify sugar as the donor substrate, the saccharide residue of the glycopeptide that can directly import on the peptide backbone, exists or be added on the saccharide residue on the peptide PEG or PPG.
The receptor of sialyltransferase is present on the peptide that will modify with the inventive method, and this receptor is naturally occurring structure or reorganization ground, zymetology ground or chemically adds.Suitable receptor comprises, galactosyl receptor for example, as Gal β 1,4GlcNAc, Gal β 1,4GalNAc, Gal β 1,3GalNAc, LNT (oligosaccharide)., Gal β 1,3GlcNAc, GalNAc, Gal β 1,3GalNAc, Gal β 1,6GlcNAc, Gal β 1,4Glc (lactose) and other receptors well known by persons skilled in the art (for example referring to, Paulson etc., J.Biol.Chem.253:5617-5624 (1978)).
In a specific embodiment, the sialyltransferase receptor promptly is present on the glycopeptide to be finished after synthesizing in the glycopeptide body.Such glycopeptide can use the inventive method do not carry out glycopeptide glycosylation pattern modification in advance and by sialylated.In addition, method of the present invention can be used for the sialylated peptide that does not comprise suitable receptor; At first make it comprise receptor by the method known to those skilled in the art modified peptides.In a kind of exemplary specific embodiment, under the effect of GalNAc transferring enzyme, add the GalNAc residue.
In an exemplary specific embodiment, by synthesizing galactosylated acceptor, for example GalNAc to the additional galactose residue of the suitable receptor that is connected with peptide.This method comprises hatches peptide to be finished and the reactant mixture that contains an amount of galactosyltransferase (for example Gal β 1,3 or Gal β 1,4) and suitable galactose donor (for example UDP-galactose).Finish with reacting fully, or cessation reaction when having added the galactose residue of scheduled volume.The method of other of the synthetic saccharide acceptor of selecting it will be apparent to those skilled in the art that.
In another specific embodiment, the oligosaccharide of " pruning " glycopeptide connection at first whole or in part, with the receptor of exposure sialyltransferase, or exposure can be added one or more suitable residues to obtain the part of suitable receptor.Enzyme (for example referring to the U.S. patent No. 5,716,812) as glycosyl transferase and endoglycosidase can be used for coupled reaction and prunes reaction.
In discussion subsequently, the modification sugar that has the water-soluble polymer of connection by utilization is illustrated method of the present invention.This discussion is clear for what illustrate, and the technical staff will understand this discussion and be equally applicable to modify the specific embodiment that sugar has therapeutic part, biomolecule or the like.
In an exemplary specific embodiment, the carbohydrate residue that " pruning " O-connects before adding modification sugar.For example the GalNAc-Gal residue is reduced to GalNAc.Modification sugar with water-soluble polymer is connected with one or more saccharide residues that expose by " pruning ".In one embodiment, " pruning " glycopeptide, add water-soluble polymer, for example Sia that is connected with water-soluble polymer, Gal or GalNAc part via glycosyl part to the O-side chain amino acid that obtains or glycopeptide polysaccharide.Modifying sugar moieties is connected with acceptor site on " pruning " glycopeptide.In addition, unaltered sugar moieties for example Gal can be added to the end that O-connects polysaccharide.
In another exemplary specific embodiment, water-soluble polymer is added on the GalNAc residue via modification sugar with galactose residue.In addition, can be added to unaltered Gal on the terminal GalNAc residue.
Yet in another example, use the sialic acid of modifying that water-soluble polymer is added on the Gal residue.
In another exemplary specific embodiment, it is the GalNAc that is connected with aminoacid that O-is connected glycosyl residue " reduction ".In one embodiment, via adding water-soluble polymer with polymer-modified Gal.Also can be added to unaltered Gal on the GalNAc, the back is the Gal with bonded water-soluble polymer.In another specific embodiment, one or more unaltered Gal residues are added on the GalNAc, the back is with water-soluble polymers decorated sialic acid part.
Use method of the present invention, may cut down and set up basically needs the carbohydrate of structure residue arbitrarily.As mentioned above, can be added to the end of carbohydrate moiety to modification sugar, or can be between peptide core and carbohydrate end.
In an exemplary example, use polymer-modified sialic acid that water-soluble polymer is added on the terminal Gal residue.Adopt suitable sialyltransferase to add the sialic acid of modifying.This method is summarized in scheme 5.
In other method, the reactive functional groups of shielding is present on the sialic acid, is summarised as scheme 6.The reactive group of shielding preferably is not used to connect the condition effect of the sialic acid and the peptide of modification.After sialic acid of modifying and peptide are covalently bound, remove and deshield, as PEG, PPG, therapeutic partly, biomolecule or other reagent connects peptide.By reacting with the maskless active group of modifying on the saccharide residue, this reagent and peptide couple together in specific mode
Scheme 6
Modify the glycosyl transferase use that sugar all can be suitable with it arbitrarily, depend on the terminal sugar (table 2) of the oligosaccharide side chain of glycopeptide.Just as discussed above, the terminal sugar of the glycopeptide that importing PEGization or PPGization structure need can be introduced during expressing natively, maybe can use the mixture of suitable glycosidase, glycosyl transferase or glycosidase and glycosyl transferase to produce after expression.
Table 2
| X=O, NH, S, CH 2, N-(R 1-5) 2. Y=X; Z=X; A=X; B=X. Q=H 2, O, S, NH, N-R. R, R 1-4=H, connexon-M, M. M=purpose part | Purpose part=acyl group-PEG; acyl group-PPG; alkyl-PEG; acyl group-alkyl-PEG; acyl group-alkyl-PEG; carbamoyl-PPG; PEG; PPG; acyl group-aryl-PEG; acyl group-aryl-PPG; aryl-PEG; aryl-PPG; Man-6-P; heparin; heparan; SLex; mannose; FGF; VFGF; protein; chrondroitin; Keratin; dermatan; albumin; integral protein; peptide etc. |
In another exemplary specific embodiment, UDP-galactose-PEG and Lac Bovis seu Bubali β 1, the reaction of 4-galactosyltransferase, thus transfer to suitable terminal N-acetyl-glucosamine structure modifying galactose.Terminal GlcNAc residue on the glycopeptide can produce during expressing, as can in the expression system as mammal, insecticide, plant or fungus, producing, and can be as required handle glycopeptide and produce by sialidase and/or glycosidase and/or glycosyl transferase.
In another exemplary specific embodiment, use GlcNAc transferring enzyme such as GNT1-5 that the GlcN of PEGization is transferred on the terminal mannose residue on the glycopeptide.In another exemplary specific embodiment, with Enzymology method N-and/or O-connection glycan structures are removed from glycopeptide, expose aminoacid or terminal saccharide residue, be connected with modification sugar subsequently.For example, remove structure that the N-of glycopeptide connects to expose the terminal GlcNAc of GlcNAc-connection-Asn on the glycopeptide with the inscribe dextranase.With UDP-Gal-PEG and suitable galactosyltransferase PEG-or PPG-galactose functional group are imported on the GlcNAc that exposes.
In another specific embodiment, utilization can directly be added to the known glycosyl transferase that saccharide residue is transferred on the peptide backbone on the peptide backbone modifying sugar.This exemplary specific embodiment is illustrated in scheme 7.The exemplary glycosyl transferase that is used for practice of the present invention includes but not limited to, GalNAc transferring enzyme (GalNAcT1-20), GlcNAc transferring enzyme, fucosyl transferase, glucotransferase, xylose transferase, mannose transferase or the like.The use of this method allows to modify on the sugared peptide that directly is added to without any carbohydrate, or is added on the glycopeptide of existence.In both cases, the interpolation of modifying sugar all occurs in the specific site that the substrate specificity by glycosyl transferase on the peptide backbone limits, rather than the interpolation at random as with chemical method proteinic peptide backbone being modified.By suitable aminoacid sequence being imported polypeptide chain, can import a series of reagent in the albumen or glycopeptide that lacks glycosyl transferase peptide substrate sequence with biotechnology.
Scheme 7
In each above-mentioned exemplary specific embodiment, after modifying sugar and peptide combining, can use one or more other chemistry or enzymatically modifying step.In a kind of exemplary specific embodiment, adopt a kind of enzyme (for example fucosyltransferase) to the terminal glycosyl unit's (for example fucose) that adds of the modification sugar that is connected in peptide.In another example, use enzymatic reaction that the site of modifying sugar and failing to connect " is added medicated cap " (for example sialylated).In addition, use chemical reaction to change the structure of the modification sugar that connects.For example, the modification of connection sugar is stablized with the key that makes it and modify the sugared peptide element that is connected or is made its unsettled reagent reacting.In another example, the element of modifying sugar with peptide in conjunction with after go protection.The technical staff will understand after modifying sugar and peptide is connected, and have a series of enzymatic and chemical method to can be used for method of the present invention.The refine of modifying sugar-peptide conjugate in addition also within the scope of the invention.
Enzyme
Glycosyl transferase
Glycosyl transferase catalytic activation sugar (donor NDP-sugar) in a step-wise fashion adds the non-reduced end of the oligosaccharide of albumen, glycopeptide, lipid or glycolipid or growth.N-connects glycopeptide and is connected oligosaccharide donor Dol-PP-NAG with lipid via transferring enzyme
2Glc
3Man
9With the global transfer synthetic, prune core then.In this case, the character of " core " sugar is different with junctional complex subsequently.Many glycosyl transferases known in the state of the art.
The glycosyl transferase that adopts among the present invention can be selected arbitrarily, modifies sugar as saccharide donor as long as it can utilize.The example of this kind of enzyme comprises Leloir path glycosyl transferase, for example galactosyltransferase, N-acetyl-glucosamine transferring enzyme, N-acetylamino galactosamine based transferase, fucosyltransferase, sialyltransferase, mannose transferase, xylose transferase, glucuronyl transferase or the like.
Synthetic for the enzymatic sugar that relates to the glycosyl transferase reaction, glycosyl transferase can clone from originating arbitrarily or separate.Many clone's glycosyl transferases and their polynucleotide sequence are known.For example referring to, " the Internet clone glycosyl transferase address correlation " (http://www.vei.co.uk/TGN/gt_guide.htm).The glycosyl transferase aminoacid sequence also can find in many public databases with the nucleotide sequence that can derive the encoding glycosyl transferring enzyme of aminoacid sequence, comprises GenBank, Swiss-Prot, EMBL or the like.
The glycosyl transferase that method of the present invention can adopt includes, but are not limited to galactosyltransferase, fucosyl transferase, glucosyltransferase, N-acetylamino galactosamine based transferase, N-acetylglucosamine transferase, glucuronyl transferase, sialyltransferase, mannose transferase, glucuronyl transferase, galacturonic acid transferring enzyme and oligosaccharide transferring enzyme.Suitable glycosyl transferase comprises the glycosyl transferase that obtains from eukaryote and prokaryote.
The DNA of encoding glycosyl transferring enzyme can be by chemosynthesis, by from suitable cell or cloned culture examination reverse transcription mRNA, obtain from the genomic library of suitable cell or the combination by these methods by examination.Examination mRNA or genomic DNA can carry out with the oligonucleotide probe from the glycosyltransferase gene sequence.Probe can be according to known and be used for method that conventional hybridization analyzes with detectable group such as fluorophor, radioactive atom or chemiluminescent group labelling.In addition, can use the PCR oligonucleotide primer that produces by the glycosyltransferase gene sequence to obtain the glycosyltransferase gene sequence by utilizing polymerase chain reaction (PCR).Referring to the U.S. patent No. 4,683,195 of Mullis etc., the U.S. patent No. 4,683,202 of Mullis.
Glycosyl transferase can be synthetic in the carrier transformed host cells of the DNA that contains the encoding glycosyl transferring enzyme.Carrier is reproducible DNA construct.Carrier is used for the DNA of amplification coding glycosyl transferase and/or expresses the DNA of encoding glycosyl transferring enzyme.Expression vector is reproducible DNA construct, and wherein the DNA sequence of encoding glycosyl transferring enzyme is connected with suitable regulating and controlling sequence operability, and this regulating and controlling sequence can influence glycosyl transferase expresses in suitable host.Need changing of this regulating and controlling sequence according to the host who selects and the method for transformation of selection.Usually, regulating and controlling sequence comprises transcripting promoter, the optional sequence of the control operon sequence of transcribing, mRNA ribosome binding site that coding is suitable, and regulates the sequence of transcribing with translation termination.Amplification vector does not need to express control domain, needed ability of just in the host, duplicating (this usually by origin of replication give with) and the selection gene that promotes the identification of transformant.
Fucosyl transferase
In some embodiments, the glycosyl transferase that is used for method of the present invention is a fucosyltransferase.The known fucosyl transferase of those skilled in the art.Exemplary fucosyl transferase comprises the enzyme of the L-fucose being transferred to the hydroxyl group sites of acceptor saccharide from the GDP-fucose.Shifting non-nucleotide sugar also can be used among the present invention to the fucosyl transferase on the receptor.
In some embodiments, acceptor saccharide is the GlcNAc in the group of Gal β (1 → 3,4) the GlcNAc beta-yl in the oligosaccharide glucosides for example.The suitable fucosyl transferase of this reaction comprises Gal β (1 → 3,4) GlcNAc β 1-α (1 → 3,4) fucosyltransferase (FTIII E.C.No.2.4.1.65), its at first from human milk, identified (referring to Palcic etc., Carbohydrate Res.190:1-11 (1989); Prieels, etc., J.Biol.Chem.256:10456-10463 (1981); And Nunez etc., Can.J.Chem.59:2086-2095 (1981)), be also included within Gal β (1 → 4) GlcNAc β-α fucosyl transferase (FTIV, FTV, FTVI) of finding in the human serum.Also identified FTVII (E.C.No.2.4.1.65), a kind of sialic acid α (2 → 3) Gal β (1 → 3) GlcNAc β fucosyltransferase.Also identified the Gal β (1 → 3 of recombinant forms, 4) GlcNAc β-α (1 → 3,4) fucosyl transferase is (referring to Dumas, Deng, Bioorg.Med.Letters 1:425-428 (1991) and Kukowska-Latallo etc., Genes and Development 4:1288-1303 (1990)).Other exemplary fucosyl transferase comprises for example α 1,2 fucosyl transferase (E.C.No.2.4.1.69).Enzymatic is fucosylated can be undertaken by the method that the Eur.J.Biochem.191:169-176 such as Mollicone (1990) or the U.S. patent No. 5,374,655 are described.The cell that is used to produce fucosyltransferase also comprises the enzymatic system of synthetic GDP-fucose.
Galactosyltransferase
Organize in the concrete scheme at another, glycosyl transferase is a galactosyltransferase.Exemplary galactosyltransferase comprises α (1,3) galactosyltransferase (E.C.No.2.4.1.151, for example referring to Dabkowski etc., Nature 345:229-233 (1990) such as Transplant Proc.25:2921 (1993) and Yamamoto, cattle (GenBank j04989, Joziasse etc., J.Biol.Chem.264:14290-14297 (1989)), Mus (GenBank m26925; Larsen etc., Proc.Nat ' l.Acad.Sci.USA86:8227-8231 (1989)), pig (GenBank L36152; Strahan etc., Immunogenetics 41:101-105 (1995)).Another suitable α 1,3 galactosyltransferase is to participate in the blood B group synthetic enzyme of antigen (EC 2.4.1.37, Yamamoto etc., J.Biol.Chem.265:1146-1151 (1990) (people)).
That be suitable for method of the present invention in addition is β (1,4) galactosyltransferase, comprise for example EC 2.4.1.90 (LacNAc synzyme) and EC 2.4.1.22 (lactose synthetase) (cattle (D ' Agostaro etc., Eur.J.Biochem.183:211-217 (1989)), people (Masri etc., Biochem.Biophys.Res.Commun.157:657-663 (1988)), Mus (Nakazawa etc., J.Biochem.104:165-168 (1988)) and E.C.2.4.1.38 and ceramide galactosyltransferase (EC 2.4.1.45, Stahl etc., J.Neurosci.Res.38:234-242 (1994)).Other suitable galactosyltransferase for example comprises, α 1,2 galactosyltransferase (from for example foxtail millet wine fragmentation sugar yeast (Schizosaccharomyces pombe), Chapell etc., Mol.Biol.Cell5:519-528 (1994)).
From cloned genes by genetic engineering production such as enzyme GalNAc T
I-XIVProtein be well-known.For example referring to, the U.S. patent No. 4,761,371.A method relates to collects enough samples, determine aminoacid sequence by the N-terminal order-checking then, then this information is used to separate the cDNA clone of (film in conjunction with) transferring enzyme of coding total length, the expression that this cDNA is cloned in insect cell line Sf9 causes the synthetic of abundant active enzyme.Using semi-quantitative analysis to determine the receptor-specific of this enzyme to the aminoacid around the known glycosylation site in 16 kinds of different protein then, is the external glycosylation research of synthetic peptide then.The verified certain amino acid residue of this work is in the excessive existence of glycosylated fragments of peptides, and the residue of the specific site around glycosylated serine and the threonine residues is renderd a service receptor and perhaps had than other amino acid moiety remarkable influence more.
Sialyltransferase
Sialyltransferase is the glycosyl transferase that is used for reconstitution cell of the present invention and reactant mixture of another kind of type.The cell that produces the recombinant sialyltransferase also produces cmp sialic acid, and it is the sialic acid donor of sialyltransferase.The example that is suitable for sialyltransferase of the present invention comprises ST3Gal III (for example rat or people ST3Gal III), ST3Gal IV, ST3GalI, ST6GalI, ST3GalV, ST6GalII, ST6GalNAcI, ST6GalNAcII and ST6GalNAcIII (the sialyltransferase nomenclature of Cai Yonging is according to Tsuji etc., the description of Glycobiology 6:v-xiv (1996)) here.A kind of exemplary α (2 that is called, 3) α (2 of sialyltransferase (EC 2.4.99.6), 3) sialyltransferase is transferred to sialic acid the non-reduced terminal Gal of Gal β 1 → 3Glc disaccharide or glucosides, referring to Van denEijnden etc., J.Biol.Chem.256:3159 (1981), Weinstein etc., J.Biol.Chem.257:13845 (1982) and Wen etc., J.Biol.Chem.267:21011 (1992).Another are exemplary 2 years old, 3-sialyltransferase (EC 2.4.99.4) is transferred to sialic acid on the non-reduced terminal Gal of disaccharide or glucosides, referring to Rearick etc., J.Biol.Chem.254:4444 (1979) and Gillespie etc., J.Biol.Chem.267:21004 (1992).Other exemplary enzyme comprises Gal-β-1,4-GlcNAc α-2,6 sialyltransferase (referring to Kurosawa etc., Eur.J.Biochem.219:375-381 (1994)).
Preferably, glycosylation for the glycopeptide carbohydrate, sialyltransferase can be transferred to sequence Gal β 1 to sialic acid, and on the 4GlcNAc-, it is the sequence modal second from the bottom (referring to table 3) under the terminal sialic acid on the sialylated fully carbohydrate structure.
Table 3: adopt Gal β 1, the 4GlcNAc sequence is as the sialyltransferase of receptor substrate
| Sialyltransferase | The source | The sequence that forms | Ref. |
| ST6Gal I | Mammal | NeuAcα2，6Galβ1，4GlCNAc- | 1 |
| ST3Gal III | Mammal | NeuAcα2，3Galβ1，4GlCNAc- NeuAcα2，3Galβ1，3GlCNAc- | 1 |
| ST3Gal IV | Mammal | NeuAcα2，3Galβ1，4GlCNAc- NeuAcα2，3Galβ1，3GlCNAc- | 1 |
| ST6Gal II | Mammal | NeuAcα2，6Galβ1，4GlCNA | |
| ST6Gal II | Photobacterium | NeuAcα2，6Galβ1，4GlCNAc- | 2 |
| ST3Gal V | N.meningitides N.gonorrhoeae | NeuAcα2，3Galβ1，4GlCNAc- | 3 |
1) Goochee etc., Bio/Technology 9:1347-1355 (1991)
2) Yamamoto etc., J.Biochem.120:104-110 (1996)
3) Gilbert etc., J.Biol.Chem.271:28271-28276 (1996)
A kind of exemplary sialyltransferase that is used for the inventive method is ST3Gal III, is also referred to as α (2,3) sialyltransferase (EC 2.4.99.6).This enzyme catalysis sialic acid is to Gal β 1,3GlcNAc or Gal β 1, and the transfer of the Gal of 4GlcNAc glucosides is (for example referring to Wen etc., J.Biol.Chem.267:21011 (1992); Van den Eijnden etc., J.Biol.Chem.256:3159 (1991)), the oligosaccharide of agedoite connection is sialylated in the responsible glycopeptide.Sialic acid is connected with Gal, forms α and connect between two sugar.Key (connection) between two sugar is between the site 3 of the site 2 of NeuAc and Gal.This special enzyme can separate (Weinstein etc., J.Biol.Chem.257:13845 (1982)) from Hepar Mus is dirty; People cDNA (Sasaki etc. (1993) J.Biol.Chem.268:22782-22787; Kitagawa ﹠amp; Paulson (1994) J.Biol.Chem.269:1394-1401) and genome (Kitagawa etc., (1996) J.Biol.Chem.271:931-938) DNA sequence be known, help by recombinant expressed production this kind of enzyme.In a preferred implementation, sialylated method of the present invention adopts rat ST3Gal III.
The sialyltransferase of the present invention that is used for that other is exemplary comprises that those from the isolating enzyme of campylobacter jejuni (Campylobacter jejuni), comprise α (2,3), for example referring to WO99/49051.
Sialyltransferase except enzyme listed in the table 3 also can be used for the sialylated economy of commercially important glycopeptide and extensive efficiently technology.As the simple test of the application of finding these other enzymes, make the different amount (1-100mU/mg albumen) of each enzyme and take off sialic acid-α
1AGP (at 1-10mg/ml) reaction, relatively the purpose sialyltransferase is with respect to the ability of the sialylated glycopeptide of cattle ST6Gal I, ST3Gal III or these two sialyltransferases.In addition, other connects the alternative sialic acid-α that takes off of oligosaccharide from glycopeptide or N-that peptide main chain enzymatic discharges
1AGP is used for this appraisal.Than ST6Gal I more effectively the N-of the sialylated glycopeptide sialyltransferase that connects oligosaccharide can be used for the extensive technology (illustrational ST3Gal III as disclosed herein) of the sialylated practicality of peptide.
Other glycosyl transferase
It will be appreciated by those skilled in the art that other glycosyl transferase can be substituted into the similar transferring enzyme cycle, as the sialyltransferase that has write up.Especially glycosyl transferase can also be a glucosyltransferase etc., for example Alg8 (Stagljov etc., Proc.Natl.Acad.Sci.USA91:5977 (1994)) or Alg5 (Heesen etc., Eur.J.Biochem.224:71 (1994)).
N-acetylamino galactosamine based transferase also is used for practice of the present invention.Suitable N-acetylamino galactosamine based transferase includes, but are not limited to α (1,3) N-acetylamino galactosamine based transferase, β (1,4) N-acetylamino galactosamine based transferase (Nagata etc., J.Biol.Chem.267:12082-12089 (1992) and Smith etc., J.Biol Chem.269:15162 (1994)) and polypeptide N-acetylamino galactosamine based transferase (Homa etc., J.Biol.Chem.268:12609 (1993)).Suitable N-acetylglucosaminyl transferase comprises GnTI (2.4.1.101, Hull etc., BBRC 176:608 (1991)), GnTII, GnTIII (Ihara etc., J.Biochem.113:692 (1993)), GnTIV and GnTV (Shoreiban etc., J.Biol.Chem.268:15381 (1993)), the N-acetylamino glucosyl transferring enzyme (Bierhuizen etc. that O-connects, Proc.Natl.Acad.Sci.USA 89:9326 (1992)), N-acetylglucosamine-1-phosphotransferase (Rajput etc., Bichem are (1992) J.285:985) and hyaluronan synthase.
Mannose transferase can be used for shifting the mannose part of modification.Suitable mannose transferase comprises α (1,2) mannose transferase, α (1,3) mannose transferase, α (1,6) mannose transferase, β (1,4) mannose transferase, Dol-P-Man synthase, OCh1 and Pmt1 (referring to Kornfeld etc., Annu.Rev.Biochem.54:631-664 (1985)).
Xylose transferase also can be used for the present invention, for example referring to, Rodgers etc., Biochem.J., 288:817-822 (1992) and Elbain etc., the U.S. patent No. 6,168,937.Other suitable glycosyl transferase circulates in the following document to be described: Ichikawa etc., JACS114:9283 (1992), Wong etc., J.Org.Chem.57:4343 (1992) and Ichikawa etc., CARBOHYDRATES AND CARBOHYDRATE POLYMERS.Yaltami, ed. (ATL Press, 1993).
Former ribosyltransferase also is used for practice of the present invention, and this glycosyl transferase comprises and relate to the synthetic enzyme of fat oligosaccharide (LOS) that it is produced by many gram negative bacteria.LOS has terminal polysaccharide sequence usually, the polysaccharide conjugates of finding in this polysaccharide sequence anthropomorphic dummy's surface epithelial cell or the host's secretions (Preston etc., Critical Reviews in Microbiology23 (3): 139-180 (1996)).This kind of enzyme includes, but are not limited to, albumen as the rfa operon of the species of escherichia coli and Salmonella typhimurium (Salmonella typhimurium), comprise β 1,6 galactosyltransferases and β 1,3 galactosyltransferases (for example referring to, EMBL accession number M80599 and M86935 (escherichia coli); EMBL accession number S56361 (Salmonella typhimurium)), glucosyltransferase (Swiss-Prot accession number P25740 (escherichia coli), β 1,2 glucosyltransferases (rfaJ) (Swiss-Prot accession number P27129 (escherichia coli) and Swiss-Prot accession number P19817 (Salmonella typhimurium)), and β (1,2)-N-acetylamino glucosyl transferring enzyme (rfaK) (EMBL accession number U00039 (escherichia coli).The known glycosyl transferase of other aminoacid sequence comprises the enzyme by the operon coding of for example rfaB, it is in for example Klebsiella pneumonia (Klebsiella pneumoniae), escherichia coli, Salmonella typhimurium, intestinal Salmonella (Salmonella enterica), yersinia enterocolitica (Yersinia enterocolitica), Mycobacteriumleprosum, and the rh1 operon of bacillus pyocyaneus (Pseudomonas aeruginosa).
Relate to producing and contain lacto-N-neotetraose, D-galactosyl-β-1,4-N-acetyl group-D-Glucoamino-β-1,3-D-galactosyl-β-1,4-D-glucose and p
kBlood group trisaccharide sequence D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose and P
kBlood type trisaccharide sequence D-galactosyl-α-1,4-D-galactosyl-β-1, the glycosyl transferase of the structure of 4-D-glucose also is applicable to the present invention, be identified among the LOS of this structure in mucosal disease substance Diplococcus gonorrhoeae (Neisseria gonnorhoeae) and Neisseria meningitidis (Scholten etc., J.Med.Microbiol.41:263-243 (1994)).The gene of biosynthetic glycosyl transferase that relates to these structures from the coding of Neisseria meningitidis and Diplococcus gonorrhoeae is from Neisseria meningitidis immunologic pattern L3 and L1 (Jennings etc., Mol.Microbiol.18:729-740 (1995)) identification (Gotshlich, J.Exp.Med.180:2181-2190 (1994)) and among the Diplococcus gonorrhoeae mutant F62.In Neisseria meningitidis, be coded in by the site of lgtA, lgtB and three genomic constitutions of lgE and add last three needed glycosyl transferases (Wakarchuk etc., J.Biol.Chem.271:19166-73 (1996)) when sugared in the lacto-N-neotetraose chain.Proved the enzymatic activity of lgtB and lgtA gene outcome recently, first positive evidence (Wakarchuk etc., J.Biol.Chem.271 (45): 28271-276 (1996)) of their glycosyl transferase function is provided.In Diplococcus gonorrhoeae, two other genes are arranged, promptly lgtD is added to β-D-GalNAc the site 3 of terminal galactose of lacto-N-neotetraose structure and the lactose element that lgtC is added to terminal α-D-Gal the LOS of shortening, thereby produces p
kBlood group antigen structure (Gotshlich (1994) sees above).In Neisseria meningitidis, other immunologic pattern L1 also expresses p
kBlood group antigen, and shown and carry lgtC gene (Jennings etc. (1995) see above).Eisseria glycosyl transferase and relevant gene are described among 553 (Gotschlich) also at USPN 5,545.From the α 1 of helicobacter pylori (Helicobacter pylori), 2-fucosyltransferase and α 1,3-fucosyl transferase gene are also identified (Martin etc., J.Biol.Chem.272:21349-21356 (1997)).The campylobacter jejuni glycosyl transferase also is used for the present invention's (for example referring to, http://afmb.cnrs-mrs.fr/～pedro/CAZY/gtf 42.html).
Sulfotransferase
The present invention also provides to produce and has comprised sulfating numerator, for example comprises the method for the peptide of sulphation polysaccharide such as heparin, Heparan sulfate, carragenen and relevant chemical compound.Suitable sulfotransferase comprises for example chrondroitin-6-sulfotransferase (the chicken cDNA that Fukuta etc. describe, J.Biol.Chem.270:18575-18580 (1995); GenBank accession number D49915).Glycosyl aminoglycan N-acetyl-glucosamine N-deacetylase/N-sulfotransferase 1 (Dixon etc., Genomics 26:239-241 (1995); UL18918), and glycosyl aminoglycan N-acetyl-glucosamine N-deacetylase/N-sulfotransferase 2 (Muridae cDNA that Orellana etc. describe, J.Biol.Chem.269:2270-2276 (1994) and Eriksson etc., J.Biol.Chem.269:10438-10443, (1994); The people cDNA that describes among the GenBank accession number U2304).
The bonded glycosyl transferase of cell
In another embodiment, the enzyme that uses in the method for the present invention is the bonded glycosyl transferase of cell.Although known many soluble sugar based transferases (for example referring to, the U.S. patent No. 5,032,519), when being associated with cell, normally film combining form of glycosyl transferase.Yan Jiu many membrane bound enzymes are considered to integral protein so far, that is to say that they do not discharge from film by supersound process, and need the detergent dissolving.Discerned surperficial glycosyl transferase, had realized that also these surperficial transferring enzymes keep catalytic activity under physiological condition at the cell surface of vertebrates and invertebrates.Yet the function of the cell surface glycosyl transferase of more identifications is the identification (Roth, MOLECULAR APPROACHES toSUPRACELLULAR PHENOMENA, 1990) between sustenticular cell.
Set up the method that changes the glycosyl transferase of cellular expression, Larsen etc. for example, Proc.Natl.Acad.Sci.USA 86:8227-8231 (1989) has reported a kind of genetic method of cDNA sequence of separating clone, to determine the expression of cell surface oligosaccharide structure and their associated sugars based transferase.With from known expression UDP-galactose: β-D-galactose-1,4-N-acetyl-D-glucose acyl ammonia α-1, the cDNA library transfection COS-1 cell that isolating mRNA generates in the murine cell line of 3-galactosyltransferase, cultivate cells transfected then, and analyze the activity of α 1-3 galactosyltransferase.
Proc.Natl.Acad.Sci.USA89:2713-2717 such as Francisco (1992) disclose a kind of method that beta-lactamase is anchored to colibacillary outer surface.By the signal sequence of (i) outer membrane protein, the (ii) transmembrane segment of outer membrane protein, and triple fusants that (iii) complete sophisticated beta-lactamase sequence is formed is produced, and causes the active lactamase molecule of surface combination.Yet the Francisco method only limits to the prokaryotic cell system, admits as the author, and finishing appropriate functional needs triple completely fusants.
Fused protein
In the other exemplary specific embodiment, method utilization of the present invention has the fused protein of the synthetic enzymatic activity of the glycopeptide conjugate that relates to needs more than.Fused polypeptide can be by for example forming with the catalytic activity territory of the bonded glycosyl transferase of catalysis active domain of auxiliary enzymes.The step that the auxiliary enzymes catalyst structure domain can catalysis forms as the nucleotide sugar of glycosyl transferase donor, or catalysis relates to the reaction in glycosyl transferase cycle.For example, the polynucleotide of encoding glycosyl transferring enzyme can be connected by reading frame with the polynucleotide that coding relates to the synthetic enzyme of nucleotide sugar.Therefore what the fusion rotein that produces not only can the catalysis nucleotide sugar is synthetic, and can the catalysis sugar moieties to the transfer of acceptor molecule.Fusion rotein can be the two or more cyclophorases that are connected with an effable nucleotide sequence.In the other specific embodiment, fusion rotein comprises the catalytic activity territory of two or more glycosyl transferases.For example referring to, 5,641,668.Utilize different suitable fused proteins can easily design and produce modification glycopeptide of the present invention (for example, referring to, PCT patent application PCT/CA98/01180 was disclosed as WO 99/31224 on June 24th, 1999).
Fixed enzyme
Except that the bonded enzyme of cell, the present invention also provides the application that is fixed on the enzyme on solid and/or the solubility holder.In a kind of exemplary specific embodiment, the glycosyl transferase that provides a kind of the method according to this invention to be connected with PEG via complete glycosyl connexon.PEG-connexon-enzyme conjugate selectively is connected with solid carrier.The application of the solid phase carrier enzyme of method of the present invention is oversimplified the progressively foundation (work up) of reactant mixture and the purification of product, and endonuclease capable is easily reclaimed.Used the glycosyl transferase conjugate in the method for the present invention.The combination of other enzyme and holder it will be apparent to those skilled in the art that.
By the glycosylation of recombinant method
Mutant human growth hormone's glycosylation can be finished in cell by recombination form.Encoding mutant body human growth hormone's (comprising that the N-of at least one new importing or O-connect glycosylation site) polynucleotide sequence can the transfection appropriate host cell be for example to derive from yeast, insecticide or mammiferous eukaryotic cell lines.Pass through the glycosylation mechanism glycosylation of host cell by the mutant human growth hormone of such cell line reorganization generation.
The purification of glycosylated mutant hGH
The glycosylated human growth hormone who produces by above-mentioned operation is purification before using preferably.Can use standard, well-known technology, for example thin layer or thick-layer chromatography, column chromatography, ion-exchange chromatography or membrane filter technique.The preferred membrane filter technique that uses more preferably utilizes reverse osmotic membrane, or one or more column chromatography technology reclaims, as discussed below and discuss in the document of quoting here.
If glycosylated mutant human growth hormone produces in the cell,, remove host cell or the segmental microgranule fragment of cytolysis by for example centrifuging or ultrafiltration as the first step; Alternatively, can concentrate membrance concentration albumen with commercially available albumen, then by one or more following method isolated polypeptide variants from other impurity that are selected from: the immunoadsorption chromatography, the ion exchange column partition method (for example diethyllaminoethyl (DEAE) contain carboxymethyl or the substrate of sulfopropyl group on), at Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S; LcA-agarose gel; WGA-agarose gel; Con A-agarose gel; Ether Toyopearl; Butyl Toyopearl; PhenylToyopearl; the chromatography method on SP-agarose gel or the protein A agarose gel; SDS-PAGE chromatography method; silicon dioxide chromatography partition method; chromatofocusing; reversed-phase HPLC (silica gel that for example has additional fat-based); gel filtration for example uses polydextran gel molecular sieve or size exclusion chromatography; optionally in conjunction with the chromatography method on the post of polypeptide and ethanol or ammonium sulfate precipitation.
The glycosylated mutant human growth hormone who produces in cultivation is usually at first by extracting from cell, cell lysates, culture medium or the like, carries out then that one or many concentrates, saltouts, aquo ion exchanges or size exclusion chromatographic step and separating.In addition, can pass through the affinity chromatography purification glycoprotein.At last, can adopt HPLC to carry out last purification step.
Protease inhibitor, for example Fumette (PMSF) can be included in any above-mentioned step, with the Profilin hydrolysis, can comprise that also antibiotic is to prevent the growth of accidental pollution thing.
Sometimes, at first use commercially available albumen to concentrate the supernatant of membrance concentration, for example use a kind of Amicon or Millipore Pellicon ultrafiltration apparatus from the system of producing glycosylated human growth hormone of the present invention.Behind the concentration step, can be applied to suitable purification substrate to concentrate.For example, suitable affinity substrate can comprise part, agglutinin or the antibody molecule that is combined in the peptide on the suitable holder.In addition, can adopt anion exchange resin, for example have substrate or basic unit that side is hung the DEAE group.Suitable substrate comprises acrylamide, agarose, glucosan, cellulose or other the type that is generally used for protein purification.Also can adopt cation-exchange step, suitable cationite comprises the various insoluble substrate that contain sulfopropyl or carboxymethyl group, preferred especially sulfopropyl group.
At last, can adopt one or more RP-HPLC steps to be further purified glycosylated mutant human growth hormone, described RP-HPLC uses hydrophobicity RP-HPLC medium for example to have the silica gel that side is hung methyl or other fat-based.Can also adopt the various combination of some or all above-mentioned purification step that glycoprotein is provided.
Of the present invention glycosylated mutant human growth hormone by large-scale fermenting and producing can be by being similar to J.Chromatog.296:171 (1984) disclosed method purification such as Urdal.This list of references has been described the RP-HPLC step of two recombinant human IL-2 purification that carry out in succession on preparation scale HPLC post.In addition, can use for example technology purification glycoprotein of affinity chromatography.
The functional analysis of mutant hGH
After glycosylated mutant human growth hormone's the production, behind the preferred purification, use the biological function of some method test glycoproteins well known in the prior art.Functional selection is with human growth hormone's the different bases that are characterized as, for example the activation and the activity in promoting the cell growth of it and the bonded specificity of growth hormone receptor, hGH receptor.In each was analyzed, wild type the human growth hormone included as positive control.
Can carry out the hGH receptor of radioreceptor binding analysis detection of radioactive labels and the combination between the mutant human growth hormone of the present invention.The detailed description of such analysis can be found in the literature, Tsushima etc. for example, J.Clin.Endocrinol.Metab., 37:334-337 (1973); Chin etc., Endocr.Meta.37:334 (1973); And the U.S. patent No. 4,871,835,5,079,230.
Promote ability (Parlow etc., the Endocrinology 77:1126 (1965) of cell growth by the method evaluation mutant human growth hormone of for example tibia test; U.S. the patent No. 4,871, and 835).In brief, excise the hypophysis of the mice in 28-30 days ages, keep and to treat in 10-14 days.Then by every day subcutaneous injection give human growth hormone's mutant with the rat recombinant sources.Slaughter animal on the 6th day, take out the foreleg Patella, and measure the width of epiphyseal plate.Also monitor the weight of these rats when on-test and before slaughtering, and the mutant human growth hormone's that accepts the injection variable concentrations every day different group is compared.
And, can show that the mutant human growth hormone is at the biological activity of cloning and cause the ability of hGH dependency tyrosine phosphorylation from people's lymphoblastoma in the IM-9 of cell surface expression growth hormone receptor cell.Other cell type as the MB-2 cell also can be suitable for the hGH functional analysis.By show the tyrosine phosphorylation level of the cell protein that is exposed to the mutant human growth hormone at the monoclonal antibody of phosphorylated tyrosine, as descriptions such as Silva, Endocrinology, 132:101 (1993) and the U.S. patent No. 6,238,915.
Pharmaceutical composition and administration
Glycosylation mutant human growth hormone with above-mentioned required oligosaccharide determinant can be as many diseases relevant with the growth hormone defective of treatment and treatment of conditions agent.Can comprise with the growth phase related disorders of mutant human growth hormone treatment of the present invention: dwarfism, child and adult's height deficiency, cachexia/muscle loss, whole-body muscle atrophy and sex chromosomal abnormality (for example Turner syndrome).Other the disease that can use mutant hGH treatment of the present invention comprises: short bowel syndrome, lipodystrophy, osteoporosis, uremia, burn, female sterility, inostosis, common diabetes, type ii diabetes, osteoarthritis, chronic obstructive pulmonary disease (COPD) and insomnia.Mutant hGH of the present invention also can be used to promote various agglutinations, for example body tissue's regeneration, osteanagenesis and wound healing or as vaccine adjuvant.Thereby the present invention also provides a kind of pharmaceutical composition, comprises the glycosylation mutant human growth hormone of effective dose, and it is according to aforesaid method production.
Pharmaceutical composition of the present invention is suitable for using in many delivery systems.Being used for suitable prescription of the present invention can be at Remington ' s Pharmaceutical Science, MackPublishing Company, and Philadelphia, PA, 17th ed. finds in (1985).To the summary of medicament delivery method referring to Langer, Science 249:1527-1533 (1990).
The pharmaceutical composition plan is by parenteral, intranasal, part, oral or surperficial (local) administration, as preventing and/or treating by subcutaneous injection, spraying suction or Transdermal absorption.Usually this pharmaceutical composition is by parenteral, for example subcutaneous or intravenous.Therefore, the invention provides the compositions of parenteral, comprise the glycosylation mutant human growth hormone who is dissolved or suspended in the acceptable carrier, the preferred water carrier is as water, buffered water, saline, PBS or the like.Said composition also can contain the detergent just like polysorbas20 and Tween 80; Stabilizing agent as mannitol, Sorbitol, sucrose and trehalose; And as the antiseptic of EDTA and metacresol.Said composition can contain the needed auxiliary substance of pharmaceutically acceptable simulation physiological condition, as pH calibration and buffer agent, tension regulator, wetting agent, detergent or the like.
These compositionss can maybe can filter sterilization by traditional sterilization technology sterilization, and the aqueous solution of acquisition can directly be packed and use or lyophilizing, and lyophilized formulations combined with aseptic aqueous carrier before administration.The pH of preparation between 3 and 11, is more preferably from 5 to 9 usually, and most preferably from 7 to 8.
Can be prevented and/or treated with the compositions that contains glycosylated mutant human growth hormone.In therapeutic is used, give to suffer the patient of disease or the disease relevant compositions, the enough treatments or of its amount to the symptom of small part prevention disease and its complication with growth hormone deficiency.The amount of enough finishing these is called as " treatment effective dose ".The effective amount of this application is depended on the seriousness of disease or disease and patient's body weight and general situation, but patient to 70kg, usually every day from about 0.1mg to about 2, the glycosylated mutant human growth hormone of 000mg, more employing every days the dosage from about 5mg to about 200mg chemical compound.
In prophylactic applications, the compositions that contains glycosylated mutant human growth hormone of the present invention given the patient who suffers from a certain disease or have ill risk easily.This amount is called as " prevention effective dose ".In this case, accurate amount also depends on patient's health status and body weight, for the patient of 70kg body weight, usually every day from about 0.1mg to about 1,000mg, more employings are every 70kg body weight from about 5mg to about 200mg.
Single or the multiple administration of compositions can be carried out with dosage level and pattern that the treatment doctor selects.In any case pharmaceutical preparation should provide a certain amount of effective treatment patient's glycosylation mutant human growth hormone of the present invention.
Only explanation as an example of the following example is provided, rather than limitation of the present invention.Those skilled in the art will easily discern and can change or revise and produce in fact similarly result's many non-key parameter.
The human growth hormone exists with different aminoacid sequences with many different hypotypes.Two kinds of forms of identifying best comprise the hGH in Placenta Hominis source, also claim GH-V (PDB P01242), and the hGH in hypophysis source, also claim growth hormone or GH-N (P01241); Referring to Fig. 1.The hGH in hypophysis source is not glycosylated, and produces as therapeutic agent in escherichia coli.The hGH (GH-V) in Placenta Hominis source has a N-glycosylation site (referring to table 4 and Fig. 1, as shown by arrows) in amino acid/11 40 sites.
Table 4. human growth hormone (GH-V) derives from Placenta Hominis; P01242 (SEQ ID NO:2)
fptiplsrlfdnamlrarrlyqlaydtyqefeeayilkeqkysflqnpqtslcfsesiptpsnrvktqqksnle
llrisllliqswlepvqllrsvfanslvygasdsnvyrhlkdleegiqtlmwrledgsprtgqifnqsyskfdt
kshnddallknygllycfrkdmdkvetflrivqcrsvegscgf ↑
The hGH (GH-N) in hypophysis source can modify at amino acid sites 140, import N-by the nucleotide sequence sudden change that makes coded polypeptide and connect glycosylation site, therefore not encoding wild type lysine (amino acid/11 40 on the GH-N peptide sequence in table 5 and Fig. 1 is abbreviated as " k ", as shown by arrows), nucleotide sequence will be at the amino acid sites 140 coding agedoites (abbreviation " n ") (referring to Fig. 2) of GH-N.
Table 5. human growth hormone (GH-N) derives from hypophysis; P01242 (SEQ ID NO:1)
fptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpqtslcfsesiptpsnreetqqksnle
llrisllliqswlepvqflrsvfanslvygasdsnvydllkdleegiqtlmgrledgsprtgqifkqtyskfdt
nshnddallknygllycfrkdmdkvetflrivqcrsvegscgf ↑
The hGH in the hypophysis source of this sudden change does not consider to be used to produce this polypeptide expression system, can be by glycosylation or puting together of glycosyl (referring to WO 03/31464, being incorporated herein by reference) herein.The hGH in the hypophysis source of this sudden change is preferably by sugared Pegylation, and wherein Polyethylene Glycol (PEG) part is connected (referring to WO03/31464, being incorporated herein by reference) with the hGH polypeptide in the hypophysis source of sudden change herein via glycosyl bond.Fig. 3 has described the sugared Pegylation that the hGH N-that produces connects the polysaccharide mutant in Sf9 insect cell or mammalian cell.The sugared Pegylation expectation of the hGH in the hypophysis source of sudden change causes the biophysical properties that improves, includes but not limited to half-life of improving, the area under curve of improvement (AUC) value, the clearance rate of reduction and the immunogenicity of reduction.
Other method is to set up O-to connect glycosylation site in the hGH polypeptide in hypophysis source.This O-connects glycosylation site can be as using GalNAcT
2Enzymes etc. make the site of the hGH polypeptide sugar Pegylation of sudden change.One or more other transferring enzymes can be used for adding polysaccharide or glycoconjugate to this site.The hGH polypeptide in the hypophysis source of sudden change is preferably by sugared Pegylation.Fig. 4 has described the sugared Pegylation that the hGH O-that produces connects the polysaccharide mutant in escherichia coli.
Embodiment 3
As what discern by the crystal structure of hGH and its receptor, the albumen ring zone of the hGH in hypophysis source is suitable for importing the sudden change (Fig. 5) of glycosylation site most.Particularly, can be to the amino acid/11-6 (FPTIPL of the hGH aminoacid sequence in encoding wild type hypophysis source; SEQ IDNO:10), aminoacid 48-52 (PQTSL; SEQ ID NO:11), aminoacid 59-64 (PTPSNR; SEQ ID NO:12), amino acid/11 33-139 (PRTGQIF; SEQ ID NO:13), amino acid/11 33-145 (PRTGQIFKQTYSK; Or amino acid/11 39-142 (FKQT SEQ ID NO:14); SEQ ID NO:15) nucleotide sequence (referring to table 5 and Fig. 1) is introduced sudden change, thereby introduces the glycosylation site that N-connects or O-connects to the hGH polypeptide that the hypophysis of the sudden change that obtains is originated.
Fig. 6 has illustrated that the O-of 6 importings connects glycosylation site, and the arrow among Fig. 6 is represented separately to connect at GH-N O-and in the polysaccharide hGH mutant O-taken place and connect glycosylated threonine residues.
Fig. 7 illustrates separately that with Fig. 8 two other GH-N O-are connected polysaccharide hGH mutant.
Embodiment 4
Present embodiment has been described and imported the aminoacid sequence sudden change that O-connects glycosylation site, i.e. serine or threonine residues in the wild type human growth hormone sequence that preferably contains the proline site or its any modification type.
1. amino terminal sudden change
In the amino terminal mutant, the N-terminal of wild type hGH is FP
2TIP
5LS; SEQ IDNO:16 is replaced by MXnTP
2TIP
5LS or MAPTSSXnP
2TIP
5LS.Preferred examples comprises:
MVTPTIPLS；SEQ ID NO：17
MQTPTIPLS；SEQ ID NO：18
MAPTSSPTIPLS；SEQ ID NO：19
MAPTSSSPTIPLS (IL-2N-end); SEQ ID NO:20
MPTTFPTIPLS；SEQ ID NO：21
MPTSSPTIPLS；SEQ ID NO：22
MPTSSSPTIPLS；SEQ ID NO：23
2. inner mutational site 1
In this class mutant, the N-terminal of wild type hGH is FP
2TIP
5LS; SEQ ID NO:24 is replaced by ZmP
2T XnBoP
5LS.Preferred sudden change comprises:
MFPTQIPLS；SEQ ID NO：25
MFPTSIPLS；SEQ ID NO：26
MFPTSSPLS；SEQ ID NO：27
MTPTQIPLS；SEQ ID NO：28
MFPTTTPLS；SEQ ID NO：29
3. inner mutational site 2
In such sudden change, use AZmJqP
37OrXnBo Δ pY substitutes P
37Aminoacid sequence on every side, i.e. AYIP
37KEQKY; SEQ ID NO:30, Z wherein, J, O, at least one among X and the B independently is selected from Thr or Ser, and Δ can comprise Lys (K), and X can be Asp (D), preferred examples comprises:
AYIP
37TQGAY；SEQ ID NO：31
AYIP
37TSSSY；SEQ ID NO：32
AQITP
37TEQKY；SEQ ID NO：33
AYIP
37TEQSY；SEQ ID NO：34
4. inner mutational site 3
In such sudden change, use LZmJqP
48OrXnBoLC substitutes P
48Aminoacid sequence on every side, i.e. LQNP
48QTSLC; SEQ ID NO:35, Z wherein, J, at least one of O and X independently is selected from Thr or Ser, and preferred examples comprises:
LQTP
48QTSLC；SEQ ID NO：36
LQNP
48TTSLC；SEQ ID NO：37
5. inner mutational site 4
In such sudden change, use SZmUsJqP
59TPOrXnBo Δ rT substitutes P
59Aminoacid sequence on every side, i.e. SESIP
59TPNREET; SEQ ID NO:38, Z wherein, J, O, B, Δ, at least one among U and the X is independently selected from Thr or Ser, and B, Δ and Z can comprise charged aminoacid, and preferred examples comprises:
SESTP
59TPNREET；SEQ ID NO：39
SSSTP
59TPNREET；SEQ ID NO：40
SESIP
59TPNTEET；SEQ ID NO：41
SESIP
59TPNTQET；SEQ ID NO：42
SESIP
59TPTQGAT；SEQ ID NO：43
SESIP
59TPTESST；SEQ ID NO：44
SQSTP
59TPNREET；SEQ ID NO：45
SQSTP
59TPNQEET；SEQ ID NO：46
SESTP
59TPTSSST；SEQ ID NO：47
6. inner mutational site 5
In such sudden change, use SZmUsJqP
89OrXnBo Δ r λ tS substitutes P
89Aminoacid sequence on every side, i.e. SWLEP
89VQFLRS; SEQ ID NO:48, Z wherein, U, J, O, at least one among B and the X is independently selected from Thr or Ser, and J and λ can comprise charged aminoacid, and preferred examples comprises:
SWLEP
89TQGLRS；SEQ ID NO：49
SWLEP
89TQGATS；SEQ ID NO：50
SSQTP
89VQFLRS；SEQ ID NO：51
SWLEP
89TSSLSS；SEQ ID NO：52
SMVTP
89VQFLRS；SEQ ID NO：53
7. inner mutational site 6
In such sudden change, use EZmUsJqP
133OrXnBo Δ r λ tF substitutes P
133Aminoacid sequence on every side, i.e. EDGSP
133RTGQIF; SEQ ID NO:54, Z wherein, U, J, O, at least one among B and the X is independently selected from Thr or Ser, and preferred examples comprises:
EDGSP
133TTGQIF；SEQ ID NO：55
EDGSP
133NTGQIF；SEQ ID NO：56
EDGSP
133TQGQIF；SEQ ID NO：57
EDGSP
133TVGQIF；SEQ ID NO：58
EDGSP
133TTTQIF；SEQ ID NO：59
EDGSP
133TSSQIF；SEQ ID NO：60
EDGSP
133TTQGIF；SEQ ID NO：61
EDGSP
133QTGQIF；SEQ ID NO：62
EDGTP
133NTGQIF；SEQ ID NO：63
EDQTP
133NTGQIF；SEQ ID NO：64
8. inner mutational site 7
In such sudden change, with GZmUsJq Δ r
140OrXnBoS substitutes P
140Aminoacid sequence on every side, i.e. GQIFK
140QTYS; SEQ ID NO:65, Z wherein, U, J, O, at least one among B and the X is independently selected from Thr or Ser, and preferred examples comprises:
GQIFN
140QTYS；SEQ ID NO：66
GQIFN
140ITYS；SEQ ID NO：67
GQIFP
140QTSS；SEQ ID NO：68
GQIFP
140TTTS；SEQ ID NO：69
GQITP
140QTYS；SEQ ID NO：70
GQIFT
140QTYS；SEQ ID NO：71
GQIST
140QTYS；SEQ ID NO：72
GQIPT
140TTYS；SEQ ID NO：73
9.C distal process becomes
In such sudden change, use VEGSCG
190PXnBoZmUsP substitutes the C-terminal amino acid sequence VEGSCG of wild type hGH
190F; SEQ ID NO:74, Z wherein, U, at least one among B and the X is independently selected from Thr or Ser, and preferred examples comprises:
VEGSCGPTTTP；SEQ ID NO：75
VEGSCGPTSSP；SEQ ID NO：76
VEGSCGPTQGAMP；SEQ ID NO：77
VEGSCGPTTIP；SEQ ID NO：78
VEGSCGPMVTP；SEQ ID NO：79
Under above-mentioned all situations, X, Z, B, Δ, J, U, O and λ are independently selected from E (glutamate, Glu), do not have electric charge aminoacid or dipeptides combination (comprising M, F, MF) or the like arbitrarily; M, n, o, p, q, r, s and t are independently selected from 0 to 3 integer.In all cases, the hGH mutant can be with or without N-terminal M et arbitrarily.Amino acid residue is numbered according to playing beginning and end change sequence, and wherein the residue of high order end is numbered 1.Modifying the unaltered amino acid whose numbering in back remains unchanged.In hGH mutant of the present invention, can there be above-mentioned sequence modification more than one.
Although the present invention is open with reference to specific embodiment, apparent those skilled in the art can design other embodiment of the present invention and variation, and do not deviate from real essence of the present invention and scope.
All patents that the application quotes, patent application and the equal integral body of other public publication are incorporated herein by reference.
Sequence table
<110>Neose Technologies，Inc.
DeFrees，Shawn
＜120〉preparation method of human growth hormone glycosylation mutants and compositions
<130>040853-01-5101WO
<150>US 60/469,114
<151>2003-05-09
<150>US 60/494,751
<151>2003-08-13
<150>US 60/495,076
<151>2003-08-14
<150>US 60/535,290
<151>2004-01-08
<160>79
<170>PatentIn version 3.2
<210>1
<211>191
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC FEATURE
＜223〉ripe human growth hormone (GH-N)
<400>1
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 PheArg 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>2
<211>191
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC_FEATURE
＜223〉ripe human growth hormone (GH-V)
<400>2
Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg
1 5 10 15
Ala Arg Arg Leu Tyr Gln Leu Ala Tyr Asp Thr Tyr Gln Glu Phe Glu
20 25 30
Glu Ala Tyr Ile Leu 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
Val Lys 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 Leu Leu Arg Ser Val
85 90 95
Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Arg
100 105 110
His Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Trp Arg Leu
115 120 125
Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Asn Gln Ser Tyr Ser
130 135 140
Lys Phe Asp Thr Lys 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>191
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC_FEATURE
＜223〉human growth hormone's mutant 1
<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 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 Thr Thr Thr 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>4
<211>194
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC_FEATURE
＜223〉human growth hormone's mutant 2 (the N-end that O-connects)
<400>4
Pro Thr Thr Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala
1 5 10 15
Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln
20 25 30
Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu
35 40 45
Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro
50 55 60
Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg
65 70 75 80
Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu
85 90 95
Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn
100 105 110
Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met
115 120 125
Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln
130 135 140
Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu
145 150 155 160
Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val
165 170 175
Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys
180 185 190
Gly Phe
<210>5
<211>191
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC_FEATURE
＜223〉human growth hormone's mutant 3
<400>5
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 Thr 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>6
<211>193
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC_FEATURE
＜223〉human growth hormone's mutant 4 (the N-end that O-connects)
<400>6
Met Val Thr Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met
1 5 10 15
Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu
20 25 30
Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln
35 40 45
Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser
50 55 60
Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile
65 70 75 80
Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg
85 90 95
Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val
100 105 110
Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly
115 120 125
Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr
130 135 140
Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys
145 150 155 160
Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu
165 170 175
Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly
180 185 190
Phe
<210>7
<211>193
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC FEATURE
＜223〉human growth hormone's mutant 5
<400>7
Met Val Thr Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met
1 5 10 15
Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu
20 25 30
Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln
35 40 45
Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser
50 55 60
Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile
65 70 75 80
Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg
85 90 95
Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val
100 105 110
Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly
115 120 125
Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr
130 135 140
Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys
145 150 155 160
Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu
165 170 175
Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly
180 185 190
Phe
<210>8
<211>192
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC_FEATURE
＜223〉human growth hormone's mutant 6
<400>8
Met Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu
1 5 10 15
Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe
20 25 30
Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn
35 40 45
Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn
50 55 60
Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser
65 70 75 80
Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser
85 90 95
Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr
100 105 110
Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg
115 120 125
Leu Glu Asp Gly Ser Pro Thr Val Gly Gln Ile Phe Lys Gln Thr Tyr
130 135 140
Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn
145 150 155 160
Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr
165 170 175
Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
180 185 190
<210>9
<211>192
<212>PRT
＜213〉homo sapiens
<220>
<221>MISC_FEATURE
＜223〉human growth hormone's mutant 7
<400>9
Met Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu
1 5 10 15
Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe
20 25 30
Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn
35 40 45
Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn
50 55 60
Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser
65 70 75 80
Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser
85 90 95
Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr
100 105 110
Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg
115 120 125
Leu Glu Asp Gly Ser Pro Thr Thr Thr Gln Ile Phe Lys Gln Thr Tyr
130 135 140
Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn
145 150 155 160
Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr
165 170 175
Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
180 185 190
<210>10
<211>6
<212>PRT
＜213〉homo sapiens
<400>10
Phe Pro Thr Ile Pro Leu
1 5
<210>11
<211>5
<212>PRT
＜213〉homo sapiens
<400>11
Pro Gln Thr Ser Leu
1 5
<210>12
<211>6
<212>PRT
＜213〉homo sapiens
<400>12
Pro Thr Pro Ser Asn Arg
1 5
<210>13
<211>7
<212>PRT
＜213〉homo sapiens
<400>13
Pro Arg Thr Gly Gln Ile Phe
1 5
<210>14
<211>13
<212>PRT
＜213〉homo sapiens
<400>14
Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys
1 5 10
<210>15
<211>4
<212>PRT
＜213〉homo sapiens
<400>15
Phe Lys Gln Thr
1
<210>16
<211>7
<212>PRT
＜213〉homo sapiens
<400>16
Phe Pro Thr Ile Pro Leu Ser
1 5
<210>17
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the terminal mutant of N-
<400>17
Met Val Thr Pro Thr Ile Pro Leu Ser
1 5
<210>18
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the terminal mutant of N-
<400>18
Met Gln Thr Pro Thr Ile Pro Leu Ser
1 5
<210>19
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the terminal mutant of N-
<400>19
Met Ala Pro Thr Ser Ser Pro Thr Ile Pro Leu Ser
1 5 10
<210>20
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the terminal mutant of N-
<400>20
Met Ala Pro Thr Ser Ser Ser Pro Thr Ile Pro Leu Ser
1 5 10
<210>21
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the terminal mutant of N-
<400>21
Met Pro Thr Thr Phe Pro Thr Ile Pro Leu Ser
1 5 10
<210>22
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the terminal mutant of N-
<400>22
Met Pro Thr Ser Ser Pro Thr Ile Pro Leu Ser
1 5 10
<210>23
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉the terminal mutant of N-
<400>23
Met Pro Thr Ser Ser Ser Pro Thr Ile Pro Leu Ser
1 5 10
<210>24
<211>7
<212>PRT
＜213〉homo sapiens
<400>24
Phe Pro Thr Ile Pro Leu Ser
1 5
<210>25
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>25
Met Phe Pro Thr Gln Ile Pro Leu Ser
1 5
<210>26
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>26
Met Phe Pro Thr Ser Ile Pro Leu Ser
1 5
<210>27
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>27
Met Phe Pro Thr Ser Ser Pro Leu Ser
1 5
<210>28
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>28
Met Thr Pro Thr Gln Ile Pro Leu Ser
1 5
<210>29
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>29
Met Phe Pro Thr Thr Thr Pro Leu Ser
1 5
<210>30
<211>9
<212>PRT
＜213〉homo sapiens
<400>30
Ala Tyr Ile Pro Lys Glu Gln Lys Tyr
1 5
<210>31
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>31
Ala Tyr Ile Pro Thr Gln Gly Ala Tyr
1 5
<210>32
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>32
Ala Tyr Ile Pro Thr Ser Ser Ser Tyr
1 5
<210>33
<211>10
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>33
Ala Gln Ile Thr Pro Thr Glu Gln Lys Tyr
1 5 10
<210>34
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>34
Ala Tyr Ile Pro Thr Glu Gln Ser Tyr
1 5
<210>35
<211>9
<212>PRT
＜213〉homo sapiens
<400>35
Leu Gln Asn Pro Gln Thr Ser Leu Cys
1 5
<210>36
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>36
Leu Gln Thr Pro Gln Thr Ser Leu Cys
1 5
<210>37
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>37
Leu Gln Asn Pro Thr Thr Ser Leu Cys
1 5
<210>38
<211>12
<212>PRT
＜213〉homo sapiens
<400>38
Ser Glu Ser Ile Pro Thr Pro Asn Arg Glu Glu Thr
1 5 10
<210>39
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>39
Ser Glu Ser Thr Pro Thr Pro Asn Arg Glu Glu Thr
1 5 10
<210>40
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>40
Ser Ser Ser Thr Pro Thr Pro Asn Arg Glu Glu Thr
1 5 10
<210>41
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>41
Ser Glu Ser Ile Pro Thr Pro Asn Thr Glu Glu Thr
1 5 10
<210>42
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>42
Ser Glu Ser Ile Pro Thr Pro Asn Thr Gln Glu Thr
1 5 10
<210>43
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>43
Ser Glu Ser Ile Pro Thr Pro Thr Gln Gly Ala Thr
1 5 10
<210>44
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>44
Ser Glu Ser Ile Pro Thr Pro Thr Glu Ser Ser Thr
1 5 10
<210>45
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>45
Ser Gln Ser Thr Pro Thr Pro Asn Arg Glu Glu Thr
1 5 10
<210>46
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>46
Ser Gln Ser Thr Pro Thr Pro Asn Gln Glu Glu Thr
1 5 10
<210>47
<211>12
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>47
Ser Glu Ser Thr Pro Thr Pro Thr Ser Ser Ser Thr
1 5 10
<210>48
<211>11
<212>PRT
＜213〉homo sapiens
<400>48
Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser
1 5 10
<210>49
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>49
Ser Trp Leu Glu Pro Thr Gln Gly Leu Arg Ser
1 5 10
<210>50
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>50
Ser Trp Leu Glu Pro Thr Gln Gly Ala Thr Ser
1 5 10
<210>51
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>51
Ser Ser Gln Thr Pro Val Gln Phe Leu Arg Ser
1 5 10
<210>52
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>52
Ser Trp Leu Glu Pro Thr Ser Ser Leu Ser Ser
1 5 10
<210>53
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>53
Ser Met Val Thr Pro Val Gln Phe Leu Arg Ser
1 5 10
<210>54
<211>11
<212>PRT
＜213〉homo sapiens
<400>54
Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe
1 5 10
<210>55
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>55
Glu Asp Gly Ser Pro Thr Thr Gly Gln Ile Phe
1 5 10
<210>56
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>56
Glu Asp Gly Ser Pro Asn Thr Gly Gln Ile Phe
1 5 10
<210>57
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>57
Glu Asp Gly Ser Pro Thr Gln Gly Gln Ile Phe
1 5 10
<210>58
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>58
Glu Asp Gly Ser Pro Thr Val Gly Gln Ile Phe
1 5 10
<210>59
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>59
Glu Asp Gly Ser Pro Thr Thr Thr Gln Ile Phe
1 5 10
<210>60
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>60
Glu Asp Gly Ser Pro Thr Ser Ser Gln Ile Phe
1 5 10
<210>61
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>61
Glu Asp Gly Ser Pro Thr Thr Gln Gly Ile Phe
1 5 10
<210>62
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>62
Glu Asp Gly Ser Pro Gln Thr Gly Gln Ile Phe
1 5 10
<210>63
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>63
Glu Asp Gly Thr Pro Asn Thr Gly Gln Ile Phe
1 5 10
<210>64
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>64
Glu Asp Gln Thr Pro Asn Thr Gly Gln Ile Phe
1 5 10
<210>65
<211>9
<212>PRT
＜213〉homo sapiens
<400>65
Gly Gln Ile Phe Lys Gln Thr Tyr Ser
1 5
<210>66
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>66
Gly Gln Ile Phe Asn Gln Thr Tyr Ser
1 5
<210>67
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>67
Gly Gln Ile Phe Asn Ile Thr Tyr Ser
1 5
<210>68
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>68
Gly Gln Ile Phe Pro Gln Thr Ser Ser
1 5
<210>69
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>69
Gly Gln Ile Phe Pro Thr Thr Thr Ser
1 5
<210>70
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>70
Gly Gln Ile Thr Pro Gln Thr Tyr Ser
1 5
<210>71
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>71
Gly Gln Ile Phe Thr Gln Thr Tyr Ser
1 5
<210>72
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>72
Gly Gln Ile Ser Thr Gln Thr Tyr Ser
1 5
<210>73
<211>9
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>73
Gly Gln Ile Pro Thr Thr Thr Tyr Ser
1 5
<210>74
<211>7
<212>PRT
＜213〉homo sapiens
<400>74
Val Glu Gly Ser Cys Gly Phe
1 5
<210>75
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>75
Val Glu Gly Ser Cys Gly Pro Thr Thr Thr Pro
1 5 10
<210>76
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>76
Val Glu Gly Ser Cys Gly Pro Thr Ser Ser Pro
1 5 10
<210>77
<211>13
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>77
Val Glu Gly Ser Cys Gly Pro Thr Gln Gly Ala Met Pro
1 5 10
<210>78
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>78
Val Glu Gly Ser Cys Gly Pro Thr Thr Ile Pro
1 5 10
<210>79
<211>11
<212>PRT
＜213〉artificial sequence
<220>
＜223〉mutant
<400>79
Val Glu Gly Ser Cys Gly Pro Met Val Thr Pro
1 5 10
Claims (42)
1. isolating nucleic acid contains encoding mutant body human growth hormone's polynucleotide sequence, and wherein the mutant human growth hormone comprises that the N-of non-existent new importing among the corresponding wild type human growth hormone connects or O-connects glycosylation site.
2. the nucleic acid of claim 1, wherein corresponding wild type human growth hormone has the aminoacid sequence of SEQ IDNO:1 or SEQ ID NO:2.
3. the nucleic acid of claim 1, the wherein new glycosylation site that imports is near proline residue.
4. the nucleic acid of claim 3, wherein proline residue is positioned at the site 2,5,37,48,59,89,113,140 or 190 of SEQ ID NO:1 or SEQID NO:2.
5. the nucleic acid of claim 1, wherein the mutant human growth hormone is contained SEQ ID NO:3,4,5,6,7,8 or 9 aminoacid sequence.
6. the nucleic acid of claim 1, wherein this mutant human growth hormone is contained the glycosylation site more than one new importing.
7. the expression of nucleic acids box that contains claim 1.
8. the cell that contains the nucleic acid of claim 1.
9. mutant human growth hormone, the N-that contains non-existent new importing in corresponding wild type human growth hormone connects or O-connects the glycosylation site.
10. the mutant human growth hormone of claim 9, wherein corresponding wild type human growth hormone has the aminoacid sequence of SEQ ID NO:1 or SEQ ID NO:2.
11. the mutant human growth hormone of claim 9, the wherein new glycosylation site that imports is near proline residue.
12. the mutant human growth hormone of claim 11, wherein proline residue is positioned at the site 2,5,37,48,59,89,113,140 or 190 of SEQID NO:1 or SEQ ID NO:2.
13. the mutant human growth hormone of claim 9 is contained SEQ ID NO:3,4,5,6,7,8 or 9 aminoacid sequence.
14. the mutant human growth hormone of claim 9, wherein this mutant human growth hormone is contained the glycosylation site more than one new importing.
15. the mutant human growth hormone of claim 9 is contained the water-soluble polymer that is connected with glycosylation site by the glycosyl connexon.
16. the mutant human growth hormone of claim 15, wherein said glycosyl connexon is complete glycosyl connexon.
17. the mutant human growth hormone of claim 15, wherein glycosylation site is the sudden change glycosylation site.
18. a method of producing the mutant human growth hormone, the N-that this mutant human growth hormone is contained non-existent new importing in corresponding wild type human growth hormone connects or O-connection glycosylation, and this method comprises the following steps:
(a) recombinant production mutant human growth hormone; With
(b) make this mutant human growth hormone in the new glycosylation site glycosylation that imports.
19. the method for claim 18, wherein corresponding wild type human growth hormone has the aminoacid sequence of SEQID NO:1 or SEQ ID NO:2.
20. the method for claim 18, the wherein new glycosylation site that imports is near proline residue.
21. the method for claim 20, wherein proline residue is positioned at the site 2,5,37,48,59,89,113,140 or 190 of SEQ ID NO:1 or SEQ ID NO:2.
22. the method for claim 18, wherein the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
23. the method for claim 18, wherein this mutant human growth hormone is contained the glycosylation site more than one new importing.
24. a pharmaceutical composition contains the mutant human growth hormone of effective dose, the N-that this mutant human growth hormone is contained non-existent new importing in corresponding wild type human growth hormone connects or O-connects glycosylation.
25. the compositions of claim 24, wherein corresponding wild type human growth hormone has the aminoacid sequence of SEQID NO:1 or SEQ ID NO:2.
26. the compositions of claim 24, the wherein new glycosylation site that imports is near proline residue.
27. the compositions of claim 26, wherein proline residue is positioned at the site 2,5,37,48,59,89,113,140 or 190 of SEQ ID NO:1 or SEQ ID N0:2.
28. the compositions of claim 24, wherein the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
29. the compositions of claim 24, wherein this mutant human growth hormone is contained the glycosylation site more than one new importing.
30. treat the method that the human growth hormone lacks among the patient for one kind, comprise to the mutant human growth hormone's of patient's effective dose step, wherein this mutant human growth hormone N-of comprising non-existent new importing among the corresponding wild type human growth hormone connects or O-connects glycosylation.
31. the method for claim 30, wherein corresponding wild type human growth hormone has the aminoacid sequence of SEQID NO:1 or SEQ ID NO:2.
32. the method for claim 30, the wherein new glycosylation site that imports is near proline residue.
33. the method for claim 32, wherein proline residue is positioned at the site 2,5,37,48,59,89,113,140 or 190 of SEQ ID NO:1 or SEQ ID NO:2.
34. the method for claim 30, wherein the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
35. the method for claim 30, wherein this mutant human growth hormone is contained the glycosylation site more than one new importing.
36. a method of producing mutant human growth hormone glycoconjugate, the N-that this mutant human growth hormone is contained non-existent new importing in corresponding wild type human growth hormone connects or O-connection glycosylation, and this method comprises the following steps:
(a) recombinant production mutant human growth hormone and
(b) make the glycosylation of this mutant human growth hormone enzymatic with the sugar of modifying at the new glycosylation site that imports.
37. the method for claim 36, wherein the sugar of modifying is modified with the member who is selected from poly-(ethylene glycol) and m-poly-(ethylene glycol).
38. the method for claim 36, wherein corresponding wild type human growth hormone has the aminoacid sequence of SEQID NO:1 or SEQ ID NO:2.
39. the method for claim 36, the wherein new glycosylation site that imports is near proline residue.
40. the method for claim 36, wherein proline residue is positioned at the site 2,5,37,48,59,89,113,140 or 190 of SEQ ID NO:1 or SEQ ID NO:2.
41. the method for claim 36, wherein the mutant human growth hormone is contained SEQ IDNO:3,4,5,6,7,8 or 9 aminoacid sequence.
42. the method for claim 36, wherein this mutant human growth hormone is contained the glycosylation site more than one new importing.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US46911403P | 2003-05-09 | 2003-05-09 | |
| US60/469,114 | 2003-05-09 | ||
| US60/494,751 | 2003-08-13 | ||
| US60/495,076 | 2003-08-14 | ||
| US60/535,290 | 2004-01-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101080238A true CN101080238A (en) | 2007-11-28 |
Family
ID=38907313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200480016847 Pending CN101080238A (en) | 2003-05-09 | 2004-05-07 | Compositions and methods for the preparation of human growth hormone glycosylation mutants |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101080238A (en) |
| ZA (2) | ZA200509864B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110448683A (en) * | 2011-12-09 | 2019-11-15 | 麦特保利药业有限公司 | The purposes of growth hormone fragment |
| CN110846327A (en) * | 2019-09-27 | 2020-02-28 | 宁夏伊品生物科技股份有限公司 | OPpA gene modified recombinant strain for producing L-tryptophan and construction method and application thereof |
| CN111157736A (en) * | 2018-11-08 | 2020-05-15 | 中国科学院大连化学物理研究所 | Human serum O glycosylation identification method based on chemoenzymatic |
-
2004
- 2004-05-07 ZA ZA200509864A patent/ZA200509864B/en unknown
- 2004-05-07 CN CN 200480016847 patent/CN101080238A/en active Pending
-
2007
- 2007-03-05 ZA ZA200701909A patent/ZA200701909B/en unknown
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110448683A (en) * | 2011-12-09 | 2019-11-15 | 麦特保利药业有限公司 | The purposes of growth hormone fragment |
| CN111157736A (en) * | 2018-11-08 | 2020-05-15 | 中国科学院大连化学物理研究所 | Human serum O glycosylation identification method based on chemoenzymatic |
| CN110846327A (en) * | 2019-09-27 | 2020-02-28 | 宁夏伊品生物科技股份有限公司 | OPpA gene modified recombinant strain for producing L-tryptophan and construction method and application thereof |
| CN110846327B (en) * | 2019-09-27 | 2022-08-26 | 宁夏伊品生物科技股份有限公司 | OPpA gene modified recombinant strain for producing L-tryptophan and construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200701909B (en) | 2009-02-25 |
| ZA200509864B (en) | 2008-02-27 |
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