WO2001055174A3 - Universal procedure for refolding recombinant proteins - Google Patents

Universal procedure for refolding recombinant proteins Download PDF

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Publication number
WO2001055174A3
WO2001055174A3 PCT/US2000/035632 US0035632W WO0155174A3 WO 2001055174 A3 WO2001055174 A3 WO 2001055174A3 US 0035632 W US0035632 W US 0035632W WO 0155174 A3 WO0155174 A3 WO 0155174A3
Authority
WO
WIPO (PCT)
Prior art keywords
solution
hrs
adjusted
inclusion bodies
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2000/035632
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French (fr)
Other versions
WO2001055174A2 (en
Inventor
Xinli Lin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oklahoma Medical Research Foundation
Original Assignee
Oklahoma Medical Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oklahoma Medical Research Foundation filed Critical Oklahoma Medical Research Foundation
Priority to DE60034707T priority Critical patent/DE60034707T2/en
Priority to EP00988440A priority patent/EP1255769B1/en
Priority to DK00988440T priority patent/DK1255769T3/en
Priority to JP2001561026A priority patent/JP3929777B2/en
Priority to AU24649/01A priority patent/AU783545B2/en
Priority to CA002397773A priority patent/CA2397773C/en
Publication of WO2001055174A2 publication Critical patent/WO2001055174A2/en
Publication of WO2001055174A3 publication Critical patent/WO2001055174A3/en
Anticipated expiration legal-status Critical
Priority to CY20071101003T priority patent/CY1106768T1/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C07K1/1136—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 reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are shown in Table I. The method has two key steps to unfold and then refold the proteins expressed in the inclusion bodies. The first step is to raise the pH of the protein solution in the presence of denaturing agents to pH greater than 9, preferably 10. The protein solution may be maintained at the elevated pH for a period of up to about 24 hours, or the pH immediately decreased slowly, in increments of about 0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or both steps used. In the preferred embodiment, purified inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mH EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM redued glutathion (GSH), 0.1 mM oxidized glutathion (GSSG), pH 10. The absorbance at 280 nm (OD280) of the protein solution is 5.0. This solution is rapidly diluted into 20 volumes of 20 mM Tris base. The resulting solution is adjusted to pH 9.0 with 1 M HC1 and is kept at 4 C for 24 hrs. The pH is adjusted to pH 8.8 and the solution is kept at 4 C for another 24 hrs. This process is repeated until the pH is adjusted to 8.0. After 24 hrs at pH8.0, the refolded proteins can be concentrated by ultrafiltration and applied to a gel filtration column for purification.
PCT/US2000/035632 2000-01-25 2000-12-28 Universal procedure for refolding recombinant proteins Ceased WO2001055174A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
DE60034707T DE60034707T2 (en) 2000-01-25 2000-12-28 GENERAL PROCEDURE FOR RECYCLING RECOMBINANT PROTEINS
EP00988440A EP1255769B1 (en) 2000-01-25 2000-12-28 Universal procedure for refolding recombinant proteins
DK00988440T DK1255769T3 (en) 2000-01-25 2000-12-28 Universal procedure for refolding recombinant proteins
JP2001561026A JP3929777B2 (en) 2000-01-25 2000-12-28 A routine procedure for refolding recombinant proteins
AU24649/01A AU783545B2 (en) 2000-01-25 2000-12-28 Universal procedure for refolding recombinant proteins
CA002397773A CA2397773C (en) 2000-01-25 2000-12-28 Universal procedure for refolding recombinant proteins
CY20071101003T CY1106768T1 (en) 2000-01-25 2007-07-30 GENERAL PROCEDURE FOR THE DEVELOPMENT OF RECOMBINANT PROTEINS

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US17783600P 2000-01-25 2000-01-25
US60/177,836 2000-01-25
US17836800P 2000-01-27 2000-01-27
US60/178,368 2000-01-27
US21030600P 2000-06-08 2000-06-08
US21029200P 2000-06-08 2000-06-08
US60/210,306 2000-06-08
US60/210,292 2000-06-08

Publications (2)

Publication Number Publication Date
WO2001055174A2 WO2001055174A2 (en) 2001-08-02
WO2001055174A3 true WO2001055174A3 (en) 2002-03-07

Family

ID=27497250

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/035632 Ceased WO2001055174A2 (en) 2000-01-25 2000-12-28 Universal procedure for refolding recombinant proteins

Country Status (12)

Country Link
US (3) US6583268B2 (en)
EP (1) EP1255769B1 (en)
JP (1) JP3929777B2 (en)
AT (1) ATE361313T1 (en)
AU (1) AU783545B2 (en)
CA (1) CA2397773C (en)
CY (1) CY1106768T1 (en)
DE (1) DE60034707T2 (en)
DK (1) DK1255769T3 (en)
ES (1) ES2287044T3 (en)
PT (1) PT1255769E (en)
WO (1) WO2001055174A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7119166B2 (en) 2000-01-25 2006-10-10 Oklahoma Medical Research Foundation Universal procedure for refolding recombinant proteins
CN108822202B (en) * 2018-02-07 2021-11-30 电子科技大学 Grass carp interleukin 21 recombinant protein and preparation method thereof

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EP1194449B1 (en) * 1999-06-28 2010-09-22 Oklahoma Medical Research Foundation Inhibitors of memapsin 2 and use thereof
US7186539B1 (en) * 2001-08-31 2007-03-06 Pharmacia & Upjohn Company Method for refolding enzymes
ATE466020T1 (en) 2001-11-09 2010-05-15 Univ Georgetown NEW ISOFORM OF VASCULAR DOTHELIAL CELL GROWTH INHIBITOR (VEGI)
US7166454B1 (en) * 2002-05-24 2007-01-23 Schering Corporation Codon-optimized β-secretase and methods of refolding and processing
CN1311076C (en) * 2003-04-16 2007-04-18 普罗特奥姆技术公司 Method for producing recombinaton urokinase
JP2004345958A (en) * 2003-05-13 2004-12-09 Universal Bio Research Co Ltd Protein refolding device and method of using protein refolding device
US20050227920A1 (en) * 2003-12-11 2005-10-13 Xinli Lin Methods for production of recombinant vascular endothelial cell growth inhibitor
EP1691764A2 (en) * 2003-12-11 2006-08-23 Proteomtech, Inc. Methods for treating cancer using vascular endothelial cell growth inhibitor vegi-192a
JP2005173484A (en) * 2003-12-15 2005-06-30 Canon Inc Image forming apparatus and process cartridge
GB0405330D0 (en) * 2004-03-10 2004-04-21 Astrazeneca Ab Enzyme and preparation method
US9756798B2 (en) 2004-11-19 2017-09-12 Patti D. Rubin Burrow filling compressed growing medium
US20060107589A1 (en) 2004-11-19 2006-05-25 Rubin Patti D Compressed growing medium
US20060130159A1 (en) * 2004-12-09 2006-06-15 Nick Masiello Method of purifying recombinant MSP 1-42 derived from Plasmodium falciparum
WO2006081171A1 (en) 2005-01-24 2006-08-03 Amgen Inc. Humanized anti-amyloid antibody
GB2446550B (en) * 2005-11-28 2010-12-08 Proteomtech Inc Methods for production of recombinant alpha1-antitrypsin
MY143274A (en) 2005-12-22 2011-04-15 Genentech Inc Recombinant production of heparin binding proteins
WO2007081534A2 (en) * 2005-12-22 2007-07-19 Pfizer Products Inc Method of producing catalytically active bace2 enzymes
EP1845103B1 (en) * 2006-04-10 2015-02-25 Boehringer Ingelheim RCV GmbH & Co KG Method for refolding a protein
KR100723531B1 (en) * 2006-06-13 2007-05-30 삼성전자주식회사 Semiconductor package substrate
CN101506222B (en) * 2006-07-14 2013-10-02 健泰科生物技术公司 Refolding of recombinant proteins
WO2008054592A2 (en) * 2006-09-29 2008-05-08 Proteomtech, Inc. Methods for production of recombinant plasminogen and plasmin polypeptides
US20100113355A1 (en) 2007-04-27 2010-05-06 Naresh Chennamsetty Novel antibody molecules and nucleic acids binding to fungal stress protein hsp90
WO2008132174A1 (en) * 2007-04-27 2008-11-06 Novartis Ag An immunoglobulin composition
US20090113791A1 (en) 2007-10-29 2009-05-07 Oms Investments, Inc. Compressed Coconut Coir Pith Granules and Methods for the Production and use Thereof
WO2009085200A2 (en) 2007-12-21 2009-07-09 Amgen Inc. Anti-amyloid antibodies and uses thereof
KR101063347B1 (en) 2009-04-06 2011-09-07 인하대학교 산학협력단 Refolding Method of Proteins Containing Disulfide Bonds Using Ionic Liquids
US20120142094A1 (en) 2009-04-08 2012-06-07 Ld Biopharma, Inc. Generating ips cells by protein transduction of recombinant potency-determining factors
US20160039868A1 (en) * 2013-03-22 2016-02-11 Biogenomics Limited Process for isolation and stabilisation of key intermediates for high efficiency refolding of recombinant proteins
US9982012B2 (en) 2013-03-29 2018-05-29 Dr. Reddy's Laboratories Limited Refolding of granulocyte colony stimulating factor
WO2016201608A1 (en) 2015-06-15 2016-12-22 张鹏 Method capable of being independently used for protein renaturation or capable of being used for preceding operations of protein renaturation
CN116445462A (en) * 2023-04-20 2023-07-18 西安麦博泰克生物科技有限公司 Purification preparation method of recombinant porcine pepsin and recombinant porcine pepsin

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7119166B2 (en) 2000-01-25 2006-10-10 Oklahoma Medical Research Foundation Universal procedure for refolding recombinant proteins
CN108822202B (en) * 2018-02-07 2021-11-30 电子科技大学 Grass carp interleukin 21 recombinant protein and preparation method thereof

Also Published As

Publication number Publication date
EP1255769A2 (en) 2002-11-13
EP1255769B1 (en) 2007-05-02
US7119166B2 (en) 2006-10-10
ES2287044T3 (en) 2007-12-16
DE60034707D1 (en) 2007-06-14
JP3929777B2 (en) 2007-06-13
CA2397773A1 (en) 2001-08-02
AU2464901A (en) 2001-08-07
US20030199676A1 (en) 2003-10-23
CA2397773C (en) 2008-09-02
AU783545B2 (en) 2005-11-10
US6583268B2 (en) 2003-06-24
JP2003523363A (en) 2003-08-05
US20060287504A1 (en) 2006-12-21
US20010044521A1 (en) 2001-11-22
PT1255769E (en) 2007-07-23
WO2001055174A2 (en) 2001-08-02
CY1106768T1 (en) 2012-05-23
DK1255769T3 (en) 2007-09-03
DE60034707T2 (en) 2008-01-17
ATE361313T1 (en) 2007-05-15

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