SI8611796B - Method for activating gentechnological prepared of heterolog,eukaryotic proteins, which contain disulphide's bridges, after their expression in the procaryotes. - Google Patents

Method for activating gentechnological prepared of heterolog,eukaryotic proteins, which contain disulphide's bridges, after their expression in the procaryotes. Download PDF

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SI8611796B
SI8611796B SI8611796A SI8611796A SI8611796B SI 8611796 B SI8611796 B SI 8611796B SI 8611796 A SI8611796 A SI 8611796A SI 8611796 A SI8611796 A SI 8611796A SI 8611796 B SI8611796 B SI 8611796B
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Stephen Fischer
Ralf Mattes
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Boehringer Mannheim Gmbh
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C—CHEMISTRY; METALLURGY
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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)

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Abstract

Method for activating non-glycosylated tissue plasminogen activator (t-PA) after its expression in prokaryotic cells comprises cell lysis; solubilisation under denaturing and reducing conditions, and reactivation under oxidising conditions in presence of reduced and oxidised glutathione (G5H, G55G). The new feature is that in the last stage is at pH 9-12 (pref. 9.5-11) with G5H and G55G concns. 0.1-20, pref. 0.2-10, mM and 0.01-3, pref. 0.5-1, mM, respectively, and with a non-denaturing concn. of the denaturing agent. Esp. the method is applied to t-PA expressed in E.coli and P. putida. The denaturing agent is pref. arginine, guanidine hydrochloride (both at 0.1-1, esp. 0.25-0.75, mM) or urea, at 0.5-4 (esp. 1-3.5) M in the last stage.

Claims (38)

© · · © © © ©ο c Ο »0 0 6 Ο © © Ο © Ο 0 © ©ο © I ©Ο ©Ο© © © © CCO ο c β ο c c ο f' ο c < r. ο © η η r. r r r c r· > ' r 1 PATENTNI ZAHTEVKI 1. Postopek za aktiviranje gentehnološko pripravljenih, heterolognih, eukariontskih proteinov, ki vsebujejo disulfidne mostove, po ekspresiji v prokariontih, z a) razklopom prokariontskih celic, b) solubiliziranjem eukarionskih proteinov pri denaturirnih in reducirnih pogojih, c) odločenjem redukcijskih/denaturirnih sredstev, d) reaktiviranjem pri oksidirnih pogojih s e) prevedbo tiolnih skupin solubiliziranih proteinov v mešane disulfide proteina in glutationa ob dodatku GSSG pri denaturirnih pogojih, f) tvorbo aktivnega proteina iz mešanih disulfidov pri koncentraciji GSH 0,5 do 5 mmol/1, pri pH vrednosti 7 do 10,5 ter pri ne-denaturirni koncentraciji denaturirnega sredstva.© · · © © © © ο c Ο »0 0 6 Ο © © Ο © Ο 0 © © ο © I © Ο © Ο © © © CCO ο c β ο c c ο f 'ο c < r. ο © η η r. r r r c r · > A method for activating genetically engineered, heterologous, eukaryotic proteins containing disulfide bridges, after expression in prokaryotes, for) cleavage of prokaryotic cells, b) solubilization of eukaryotic proteins in denaturing orders and denaturing orders. d) reactivation under oxidizing conditions, se) conversion of thiol groups of solubilized proteins to mixed protein and glutathione disulfides with the addition of GSSG under denaturing conditions, f) formation of active protein from mixed disulfides at a GSH concentration of 0.5 to 5 mmol / l, pH values 7 to 10.5 and at a non-denaturing concentration of denaturing agent. 2. Postopek po zahtevku 1, označen s tem, da izvedemo ekspresijo v E. coli ali P. putida.Process according to Claim 1, characterized in that the expression is carried out in E. coli or P. putida. 3. Postopek po zahtevku 1 ali 2, označen s tem, da v stopnji reaktiviranja uporabimo kot denaturirno sredstvo arginin, gvanidin hidroklorid in/ali vsaj eno spojino s splošno formulo R2-CO-NRR, (I), v kateri R in Rj pomenita vodik ali alkil z 1 do 4 atomi ogljika, R2 pa vodik ali NHR, ali alkil z 1 do 3 atomi ogljika.Process according to Claim 1 or 2, characterized in that in the reactivation step arginine, guanidine hydrochloride and / or at least one compound of the general formula R 2 -CO-NRR, (I) in which R and R 1 represent hydrogen or alkyl having 1 to 4 carbon atoms and R2 hydrogen or NHR, or alkyl having 1 to 3 carbon atoms. 4. Postopek po zahtevku 3, označen s tem, da znaša koncentracija arginina in/ali gvanidinhidroklorida 0,1 do 1,0 mol/1, zlasti 0,25 do 0,8 mol/1.Process according to Claim 3, characterized in that the concentration of arginine and / or guanidine hydrochloride is 0.1 to 1.0 mol / l, in particular 0.25 to 0.8 mol / l. 5. Postopek po zahtevku 3, označen s tem, da znaša koncentracija spojine s splošno formulo I 0,5 do 4 mol/1. zlasti 1 do 3,5 mol/1.Process according to Claim 3, characterized in that the concentration of the compound of general formula I is 0.5 to 4 mol / l. in particular 1 to 3.5 mol / l. 6. Postopek po enem izmed prejšnjih zahtevkov, označen s tem, da delamo pri stopnji reaktiviranja v prisotnosti neproteolitično učinkovitega proteina, zlasti v prisotnosti albumina iz govejega seruma.Process according to one of the preceding claims, characterized in that the reactivation step is carried out in the presence of a non-proteolytically effective protein, in particular in the presence of bovine serum albumin. 7. Postopek po enem izmed prejšnjih zahtevkov, označen s tem, da izvedemo celični razklop s pomočjo ultra zvoka, visokotlačne disperzije ali lizocima.Process according to one of the preceding claims, characterized in that the cell decomposition is carried out by means of ultrasound, high-pressure dispersion or lysozyme. 8. Postopek po zahtevku 7, označen s tem, da izvedemo razklop v razredčeni vodni pufrski raztopini, zlasti v 0,1 mol/1 Tris, pri nevtralni do šibko kisli pH vrednosti. ο ο 00 00 0 ο Ο Ο (' 0 0 ο ο 00 00 0 ο Ο Ο (' 0 0Process according to Claim 7, characterized in that the digestion is carried out in dilute aqueous buffer solution, in particular in 0.1 mol / l Tris, at neutral to weakly acidic pH. ο ο 00 00 0 ο Ο Ο ('0 0 ο ο 00 00 0 ο Ο Ο (' 0 0 © c c © ooco ο c η f' r C Ο © Ο C © crr r r r r··- crr· r f? r c r r <- r r. r, f r r r r <· > 2© c c © ooco ο c η f 'r C Ο © Ο C © crr r r r r ·· - crr · r f? r c r r < - r r. r, f r r r r < · > 2 9. Postopek po enem izmed prejšnjih zahtevkov, označen s tem, da po celičnem razklopu odločimo netopne sestavine.Process according to one of the preceding claims, characterized in that insoluble components are determined after cell digestion. 10. Postopek po enem izmed prejšnjih zahtevkov, označen s tem, da pri stopnji solubiliziranja delamo pri alkalni pH vrednosti v prisotnosti redukcijskega sredstva iz merkapto skupine ter v prisotnosti denaturirnega sredstva.Process according to one of the preceding claims, characterized in that the solubilization step is carried out at an alkaline pH value in the presence of a reducing agent from the mercapto group and in the presence of a denaturing agent. 11. Postopek po zahtevku 10, označen s tem, da delamo v prisotnosti gvanidinhidrok-lorida in/ali spojine s splošno formulo I kot denaturirnega sredstva.Process according to Claim 10, characterized in that it is carried out in the presence of guanidine hydrochloride and / or a compound of general formula I as a denaturing agent. 12. Postopek po zahtevku 11, označen s tem, da znaša koncentracija gvanidinhidrok-lorida 6 mol/1, koncentracija spojine s splošno formulo I pa 8 mol/1.Process according to Claim 11, characterized in that the concentration of guanidine hydrochloride is 6 mol / l and the concentration of the compound of general formula I is 8 mol / l. 13. Postopek po enem izmed zahtevkov 10 do 12, označen s tem, da delamo v prisotnosti DTE, /3-merkaptoetanola, cisteina ali GSH.Process according to one of Claims 10 to 12, characterized in that it is carried out in the presence of DTE, β-mercaptoethanol, cysteine or GSH. 14. Postopek po enem izmed prejšnjih zahtevkov, označen s tem, da izvedemo čiščenje in odločenje redukcijskih, oksidacijskih ali denaturirnih sredstev s pomočjo sterične izločevalne kromatografije ali dialize.Process according to one of the preceding claims, characterized in that the cleaning and separation of reducing, oxidizing or denaturing agents is carried out by means of steric separation chromatography or dialysis. 15. Postopek po enem izmed prejšnjih zahtevkov, označen s tem, da po stopnji reak-tiviranja izvedemo čistilno stopnjo s pomočjo dialize.Process according to one of the preceding claims, characterized in that after the reactivation step, a purification step is carried out by means of dialysis. 16. Postopek po enem izmed zahtevkov 1 do 15, označen s tem, da kot gentehnološko pripravljen eukariontski protein uporabimo t-PA.Process according to one of Claims 1 to 15, characterized in that t-PA is used as the genetically prepared eukaryotic protein. 17. Stimuliranja sposoben neglikoziliran tPA, ki se ga da dobiti po postopku po enem izmed zahtevkov 1 do 16.Stimulant non-glycosylated tPA obtainable by a process according to one of claims 1 to 16. 18. Postopek po zahtevku 6, označen s tem, da odločimo mešani disulfid proteina in glutationa z ionsko izmenjevalno obdelavo od nemodificiranega proteina.Process according to Claim 6, characterized in that the mixed disulfide of the protein and glutathione is separated from the unmodified protein by ion exchange treatment. 19. Postopek za aktiviranje gentehnološko prirpavljenih, heterolognih, eukariontskih proteinov, ki vsebujejo disulfidne mostove, po ekspresiji v prokariontih s celičnim razklopom, solubiliziranjem ob denaturirnih in reducirnih pogojih ter reaktivirajem ob oksidirnih pogojih v prisotnosti GSH/GSSG, označen s tem, da pri stopnji reak- ©Ο ©Ο Ο© ©© f> © ¢-,0 0 019. A method for activating genetically engineered, heterologous, eukaryotic proteins containing disulfide bridges, after expression in prokaryotes by cell digestion, solubilization under denaturing and reducing conditions, and reactivation under oxidizing conditions in the presence of GSH / H reak- © Ο © Ο Ο © ©© f > © ¢ -, 0 0 0 eecc r e e © c r o r c c © c r r © en r c- r c c- c r < r r f r- r r·- ' * c «· r r , , 3 tiviranja delamo s pH vrednostjo 8 do 12, koncentracijo GSH 0,1 do 20 mmol/1, koncentracijo GSSG 0,01 do 3 mmol/1 ter ne-denaturirno koncentracijo denaturirnega sredstva ter da v stopnji reaktiviranja uporabimo kot denaturirno sredstvo arginin in/ali vsaj eno spojino s splošno formulo R2-CO-NRRj (I), v kateri R in Rj pomenita vodik ali alkil z 1 do 4 atomi ogljika, R2 pa vodik ali NHR( ali alkil z 1 do 3 atomi ogljika, pod pogojem, da R in R, nista istočasno vodik.eecc r e e © c r o r c c © c r r © en r c- r c c- c r < rrf r- rr · - '* c «· rr,, 3 titrations are performed with a pH value of 8 to 12, a GSH concentration of 0.1 to 20 mmol / l, a GSSG concentration of 0.01 to 3 mmol / l, and a non-denaturing concentration. denaturing agent and that in the reactivation step arginine and / or at least one compound of the general formula R2-CO-NRRj (I) is used as the denaturing agent, in which R and Rj are hydrogen or alkyl of 1 to 4 carbon atoms and R2 is hydrogen or NHR (or alkyl of 1 to 3 carbon atoms, provided that R and R are not simultaneously hydrogen. 20. Postopek po zahtevku 19, označen s tem, da v stopnji reaktiviranja znaša pH vrednost 9,5 do 11.Process according to Claim 19, characterized in that the pH is 9.5 to 11 in the reactivation step. 21. Postopek po enem izmed zahtevkov 19 in 20, označen s tem, da v stopnji rak-tiviranja znaša koncentracija GSH 0,2 do 10 mmol/1 in/ali koncentracija GSSG 0,05 do 1 mmol/1.Process according to one of Claims 19 and 20, characterized in that the concentration of GSH in the reactivation step is 0.2 to 10 mmol / l and / or the concentration of GSHG is 0.05 to 1 mmol / l. 22. Postopek po enem izmed zahtevkov 19-21, označen s tem, da po solubiliziranju in pred reaktiviranjem izvedemo stopnjo čiščenja.Process according to one of Claims 19 to 21, characterized in that a purification step is carried out after solubilization and before reactivation. 23. Postopek po enem izmed zahtevkov 19-21, označen s tein, da izvedemo reak-tiviranje brez predhodnega odločenja denaturirno/redukcijskega sredstva, pri čemer reakcijsko raztopino po denaturiranju/redukciji razredčimo z reaktivirnim pufrom ter pri naslednjem reaktiviranju koncentracija GSSG prekorači preostalo rezidualno koncentracijo DTE.Process according to one of Claims 19 to 21, characterized in that the reactivation is carried out without prior decision of the denaturing / reducing agent, the reaction solution being diluted with reactivating buffer after denaturing / reduction and the GSSG concentration exceeding the residual residual concentration on subsequent reactivation. DTE. 24. Postopek po enem izmed zahtevkov 19-23, označen s tem, da izvedemo ekspresijo v E. coli ali P.putida.Process according to one of Claims 19 to 23, characterized in that it is expressed in E. coli or P. putida. 25. Postopek po enem izmed zahtevkov 19-24, označen s tem, da znaša koncentracija arginina 0,1 do 1,0 mol/1, zlasti 0,25 do 0,8 mol/1.Process according to one of Claims 19 to 24, characterized in that the concentration of arginine is 0.1 to 1.0 mol / l, in particular 0.25 to 0.8 mol / l. 26. Postopek po enem od zahtevkov 19-24, označen s tem, da znaša koncentracija spojine s splošno formulo I 0,5 do 4 mol/1, zlasti 1 do 3,5 mol/1.Process according to one of Claims 19 to 24, characterized in that the concentration of the compound of the general formula I is 0.5 to 4 mol / l, in particular 1 to 3.5 mol / l. 27. Postopek po enem izmed zahtevkov 19-26, označen s tem, da delamo pri stopnji reaktiviranja v prisotnosti neproteolitično učinkovitega proteina, zlasti v prisotnosti albumina iz govejega seruma.Process according to one of Claims 19 to 26, characterized in that the reactivation step is carried out in the presence of a non-proteolytically effective protein, in particular in the presence of bovine serum albumin. 28. Postopek po enem izmed zahtevkov 19-27, označen s tem, da izvedemo celični ο© ©O ·© ©© ©ο ο·ΦΟ OCOO © Ο © © ©ο ΟMethod according to one of Claims 19 to 27, characterized in that the cellular ο © © O · © ©© © ο ο · ΦΟ OCOO © Ο © © © ο Ο ©Ο © Ο Φ Ο c © ©ο© ο © c η ο ο © οο ^ Λ r r ο Ο C '· © r> r r ¢: 4 razklop s pomočjo ultra zvoka, visokotlačne disperzije ali lizocima.© Ο © Ο Φ Ο c © © ο © ο © c η ο ο © οο ^ Λ r r ο Ο C '· © r > r r ¢: 4 digestion by ultrasonic, high-pressure dispersion or lysozyme. 29. Postopek po zahtevku 28, označen s tem, da izvedemo razklop v razredčeni vodni pufrski raztopini, zlasti v 0,1 mol/l Tris, pri nevtralni do šibko kisli pH vrednosti.Process according to Claim 28, characterized in that the digestion is carried out in dilute aqueous buffer solution, in particular in 0.1 mol / l Tris, at a neutral to weakly acidic pH value. 30. Postopek po enem izmed zahtevkov 19-29, označen s tem, da po celičnem razklopu odločimo netopne sestavine.Process according to one of Claims 19 to 29, characterized in that insoluble components are selected after cell digestion. 31. Postopek po enem izmed zahtevkov 19-30, označen s tem, da pri stopnji solubiliziranja delamo pri alkalni pH vrednosti v prisotnosti redukcijskega sredstva iz merkapto skupine ter v prisotnosti denaturirnega sredstva.Process according to one of Claims 19 to 30, characterized in that the solubilization step is carried out at an alkaline pH value in the presence of a reducing agent from the mercapto group and in the presence of a denaturing agent. 32. Postopek po zahtevku 31, označen s tem, da delamo v prisotnosti spojine s splošno formulo I kot denaturirnega sredstva.Process according to Claim 31, characterized in that it is carried out in the presence of a compound of general formula I as a denaturing agent. 33. Postopek po zahtevku 32, označen s tem, da znaša koncentracija spojine s splošno formulo Ϊ 8 mol/l.Process according to Claim 32, characterized in that the concentration of the compound of general formula is Ϊ 8 mol / l. 34. Postopek po enem izmed zahtevkov 31 do 33, označen s tem, da delamo v prisotnosti DTE, /3-merkaptoetanola, cisteina ali GSH.Process according to one of Claims 31 to 33, characterized in that it is carried out in the presence of DTE, .beta.-mercaptoethanol, cysteine or GSH. 35. Postopek po enem izmed zahtevkov 19-34, označen s tem, da izvedemo čiščenje in odločenje redukcijskih, oksidacijskih ali denaturirnih sredstev s pomočjo sterične izločevalne kromatografije ali dialize.Process according to one of Claims 19 to 34, characterized in that the cleaning and separation of the reducing, oxidizing or denaturing agents is carried out by means of steric separation chromatography or dialysis. 36. Postopek po enem izmed zahtevkov 19-35, označen s tem, da po stopnji reak-tiviranja izvedemo čistilno stopnjo s pomočjo dialize.Process according to one of Claims 19 to 35, characterized in that a purification step is carried out by dialysis after the reactivation step. 37. Postopek po enem izmed zahtevkov 19-36, označen s tem, da kot gentehnološko pripravljen eukariontski protein uporabimo t-PA.Process according to one of Claims 19 to 36, characterized in that t-PA is used as the genetically prepared eukaryotic protein. 38. Postopek po enem izmed zahtevkov 19-36, označen s tem, da kot gentehnološko pripravljen eukariontski protein uporabimo inteferon-/3.Process according to one of Claims 19 to 36, characterized in that interferon-β is used as the genetically prepared eukaryotic protein.
SI8611796A 1985-10-23 1986-10-21 Method for activating gentechnological prepared of heterolog,eukaryotic proteins, which contain disulphide's bridges, after their expression in the procaryotes. SI8611796B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19853537708 DE3537708A1 (en) 1985-10-23 1985-10-23 METHOD FOR ACTIVATING T-PA AFTER EXPRESSION IN PROKARYONTS
YU179686A YU47185B (en) 1985-10-23 1986-10-21 PROCEDURE FOR THE ACTIVATION OF HETEROLOGICAL EUKARIOTIC PROTEINS PREPARED BY GEN-TECHNOLOGY HAVING DISULFID BRIDES OF POLSE EXPRESSION IN PROKARIOTS

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SI8611796A SI8611796A (en) 1996-10-31
SI8611796B true SI8611796B (en) 1998-06-30

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Families Citing this family (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4766205A (en) * 1985-11-13 1988-08-23 Beatrice Companies, Inc. Method for isolation of recombinant polypeptides in biologically active forms
JP2581668B2 (en) * 1985-11-27 1997-02-12 三井東圧化学株式会社 Novel DNA sequence encoding human tissue plasminogen activator derived from normal human cells and vectors and cells containing the same
US4777043A (en) * 1985-12-17 1988-10-11 Genentech, Inc. Stabilized human tissue plasminogen activator compositions
AU621051B2 (en) * 1987-04-28 1992-03-05 Amgen, Inc. Method for purifying granulocyte-macrophage colony stimulating factor
DE3722082A1 (en) * 1987-07-03 1989-01-12 Behringwerke Ag METHOD FOR DETERMINING THE ACTIVITY OF SERINE PROTEASES OR SERINE PROTEASE INHIBITORS
CA1340586C (en) * 1988-09-23 1999-06-08 Cetus Corporation Process for recovering microbially produced interferon-beta
DE3832898A1 (en) * 1988-09-28 1990-04-12 Boehringer Mannheim Gmbh PRAEPARATE OF EXPRESSED PLASMINOGEN ACTIVATOR IN PROKARYONS
DE3835350A1 (en) * 1988-10-17 1990-04-19 Boehringer Mannheim Gmbh ACTIVATION OF GENETICALLY MANUFACTURED ANTIBODY EXPRESSED IN PROKARYONS
DE3903581A1 (en) * 1989-02-07 1990-08-16 Boehringer Mannheim Gmbh FABRIC PLASMINOGEN ACTIVATOR DERIVATIVE
DE3942143A1 (en) * 1989-12-20 1991-06-27 Boehringer Mannheim Gmbh T-PA PRO STABILIZATION
EP0545999B1 (en) * 1990-08-20 1997-06-04 Novo Nordisk A/S Process for the preparation of biologically active IGF-1 using IGF-1 having an amino-terminal extension
ES2119779T3 (en) * 1990-09-05 1998-10-16 Southern Cross Biotech Pty Ltd SOLUBILIZATION OF PROTEINS IN ACTIVE FORMS.
DE4037196A1 (en) * 1990-11-22 1992-05-27 Boehringer Mannheim Gmbh METHOD FOR REACTIVATING DENATURED PROTEIN
DE4113750A1 (en) 1991-04-26 1992-10-29 Boehringer Mannheim Gmbh IMPROVEMENT OF RENATURATION IN THE SECRETION OF DISULFID-BRIDGED PROTEINS
DE4139000A1 (en) * 1991-11-27 1993-06-03 Boehringer Mannheim Gmbh METHOD OF GENERATING BIOLOGICALLY ACTIVE SS-NGF
US5212091A (en) * 1992-03-02 1993-05-18 Monsanto Company Method of producing tissue factor pathway inhibitor
AU3812993A (en) * 1992-03-24 1993-10-21 Synergen, Inc. Refolding and purification of insulin-like growth factor I
DK0600372T3 (en) 1992-12-02 1997-08-11 Hoechst Ag Process for the preparation of proinsulin with properly connected cystine bridges.
DE4405179A1 (en) * 1994-02-18 1995-08-24 Hoechst Ag Method of obtaining insulin with correctly connected cystine bridges
FR2729972B1 (en) * 1995-01-31 1997-04-18 Sanofi Sa PROCESS FOR THE EXTRACTION OF PERIPLASMIC PROTEINS FROM PROKARYOTIC MICROORGANISMS IN THE PRESENCE OF ARGININ
US5714371A (en) * 1995-05-12 1998-02-03 Schering Corporation Method for refolding insoluble aggregates of hepatitis C virus protease
US5728804A (en) * 1995-06-02 1998-03-17 Research Corporation Technologies, Inc. Use of cyclodextrins for protein renaturation
EP0904355B1 (en) * 1996-06-11 2004-03-03 Boehringer Mannheim Gmbh Method of activating denatured protein
US7153943B2 (en) 1997-07-14 2006-12-26 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins, and methods of use thereof
US6653098B1 (en) * 1998-02-23 2003-11-25 G. D. Searle & Co. Method of producing mouse and human endostatin
DE19850429A1 (en) * 1998-10-27 2000-05-04 Andre Schrattenholz Fragments
EP1048732A1 (en) * 1999-04-26 2000-11-02 F. Hoffmann-La Roche Ag Process for producing natural folded and secreted proteins
EP1077263A1 (en) * 1999-07-29 2001-02-21 F.Hoffmann-La Roche Ag Process for producing natural folded and secreted proteins by co-secretion of chaperones
IL152804A0 (en) 2000-05-16 2003-06-24 Bolder Biotechnology Inc Methods for refolding proteins containing free cysteine residues
DE10105911A1 (en) 2001-02-09 2002-08-14 Roche Diagnostics Gmbh Expression of the recombinant proteinase K from Tritirachium album in yeast
DE10105912A1 (en) * 2001-02-09 2002-08-14 Roche Diagnostics Gmbh Recombinant Proteinase K
DE102005033250A1 (en) 2005-07-15 2007-01-18 Bioceuticals Arzneimittel Ag Process for purifying G-CSF
DE202006020194U1 (en) 2006-03-01 2007-12-06 Bioceuticals Arzneimittel Ag G-CSF liquid formulation
TWI476207B (en) 2006-07-14 2015-03-11 Genentech Inc Refolding of recombinant proteins
WO2008076933A2 (en) 2006-12-14 2008-06-26 Bolder Biotechnology, Inc. Long acting proteins and peptides and methods of making and using the same
WO2011113601A1 (en) 2010-03-17 2011-09-22 Biogenerix Ag Method for obtaining biologically active recombinant human g-csf
US20130273607A1 (en) * 2010-10-20 2013-10-17 Medimmune, Llc Methods for processing inclusion bodies
HUP1200171A1 (en) 2012-03-19 2013-09-30 Richter Gedeon Nyrt Methods for the production of polypeptides
HUP1200172A2 (en) 2012-03-19 2013-10-28 Richter Gedeon Nyrt Methods for refolding g-csf from inclusion bodies
CN103852527B (en) * 2012-12-05 2015-05-13 中国科学院大连化学物理研究所 High-flux protein sample pre-treatment device
US10457716B2 (en) 2014-08-06 2019-10-29 University Of Notre Dame Du Lac Protein folding and methods of using same
WO2018121703A1 (en) 2016-12-30 2018-07-05 Biogend Therapeutics Co., Ltd. Recombinant polypeptides, compositions, and methods thereof
EP4263568A1 (en) 2020-12-18 2023-10-25 Richter Gedeon Nyrt. Methods for the purification of refolded fc-peptide fusion protein

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5135481A (en) * 1974-09-18 1976-03-25 Fujiwa Kako Kk KOJUNDOHITOROKINAAZE NO SEIHO
US4468633A (en) 1982-04-28 1984-08-28 The Bendix Corporation Adjustable microwave power combiner for a plurality of coaxially mounted impatt diodes
IL68561A (en) 1982-05-05 1991-01-31 Genentech Inc Preparation of polypeptide with human tissue plasminogen activator function,processes for making it,and dna and transformed cell intermediates thereof
US4432895A (en) * 1982-11-24 1984-02-21 Hoffmann-La Roche Inc. Monomeric interferons
GR79124B (en) * 1982-12-22 1984-10-02 Genentech Inc
GB2138004B (en) * 1983-03-25 1987-05-13 Celltech Ltd A process for the production of a protein
JPS6051119A (en) * 1983-08-30 1985-03-22 Green Cross Corp:The Dried pharmaceutical preparation of urokinase
US4530787A (en) * 1984-03-28 1985-07-23 Cetus Corporation Controlled oxidation of microbially produced cysteine-containing proteins
US4748234A (en) * 1985-06-26 1988-05-31 Cetus Corporation Process for recovering refractile bodies containing heterologous proteins from microbial hosts
US4766205A (en) * 1985-11-13 1988-08-23 Beatrice Companies, Inc. Method for isolation of recombinant polypeptides in biologically active forms
FR2596360B1 (en) * 1986-04-01 1989-02-17 Sotralentz Sa CONTAINER ON PALLET WITH FOLDED AND REINFORCED MESH PROTECTION DEVICE
JPH0651119A (en) * 1992-07-28 1994-02-25 Sekisui Chem Co Ltd Production of phase difference plate

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