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1. WO2001055174 - UNIVERSAL PROCEDURE FOR REFOLDING RECOMBINANT PROTEINS

Publication Number WO/2001/055174
Publication Date 02.08.2001
International Application No. PCT/US2000/035632
International Filing Date 28.12.2000
Chapter 2 Demand Filed 22.08.2001
IPC
A61K 38/00 2006.01
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
38Medicinal preparations containing peptides
C07K 1/113 2006.01
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
1General processes for the preparation of peptides
107by chemical modification of precursor peptides
113without change of the primary structure
C07K 5/113 2006.01
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
5Peptides having up to four amino acids in a fully defined sequence; Derivatives thereof
04containing only normal peptide links
10Tetrapeptides
113the side chain of the first amino acid containing more carboxyl groups than amino groups, or derivatives thereof, e.g. Asp, Glu, Asn
C12N 9/64 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
14Hydrolases (3.)
48acting on peptide bonds, e.g. thromboplastin, leucine aminopeptidase (3.4)
50Proteinases
64derived from animal tissue, e.g. rennin
CPC
A61K 38/00
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
38Medicinal preparations containing peptides
A61K 39/00
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
39Medicinal preparations containing antigens or antibodies
C07K 1/1133
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
1General methods for the preparation of peptides ; , i.e. processes for the organic chemical preparation of peptides or proteins of any length
107by chemical modification of precursor peptides
113without change of the primary structure
1133by redox-reactions involving cystein/cystin side chains
C07K 1/1136
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
1General methods for the preparation of peptides ; , i.e. processes for the organic chemical preparation of peptides or proteins of any length
107by chemical modification of precursor peptides
113without change of the primary structure
1136by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
C07K 5/1021
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
5Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
04containing only normal peptide links
10Tetrapeptides
1021with the first amino acid being acidic
C12N 9/6421
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9Enzymes; Proenzymes; Compositions thereof
14Hydrolases (3)
48acting on peptide bonds (3.4)
50Proteinases ; , e.g. Endopeptidases (3.4.21-3.4.25)
64derived from animal tissue
6421from mammals
Applicants
  • OKLAHOMA MEDICAL RESEARCH FOUNDATION [US]/[US]
Inventors
  • LIN, Xinli
Agents
  • PABST, Patrea, L.
Priority Data
60/177,83625.01.2000US
60/178,36827.01.2000US
60/210,29208.06.2000US
60/210,30608.06.2000US
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) UNIVERSAL PROCEDURE FOR REFOLDING RECOMBINANT PROTEINS
(FR) PROCEDURE UNIVERSELLE DE REPLIEMENT DE PROTEINES DE RECOMBINAISON
Abstract
(EN)
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.
(FR)
L'invention concerne un procédé de repliement universel qui s'avère efficace pour le repliement d'une variété de protéines très différentes exprimées dans des bactéries en tant que corps d'inclusion. Des protéines représentatives pouvant être dissoutes et repliées dans une forme active sur le plan biologique, avec la structure native, sont représentées à la Table 1. Le procédé comprend deux étapes clefs permettant de déplier et puis de replier les protéines exprimées dans les corps d'inclusion. La première étape consiste à élever le pH de la solution protéique en présence d'agents de dénaturation à un pH supérieur à 9, de préférence à 10. La solution protéique peut être maintenue à un pH élevé pendant un laps de temps allant jusqu'à 24 heures, ou le pH décroît immédiatement de manière lente, à raison d'environ 0,2 unité de pH/24heures, jusqu'au moment où la solution atteint un pH égal environ à 8,0, en variante les deux étapes peuvent être utilisées. Dans un mode de réalisation préféré, des corps d'inclusion purifiés sont dissous dans 8 M urea, 0,1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM bêta-mercaptoéthanol, 10 mM dithiothreitol (DTT), 1 mM glutathion (GSH), 0,1 mM glutathion oxydé (GSSG), pH 10. L'absorbance à 280 nm (OD280) de la solution protéique est égale a 5.0. Cette solution se dilue rapidement dans 20 volumes de 20 mM de base Tris. La solution obtenue est ajustée à pH 9.0 avec 1 M HCI et est maintenue à 4 C° pendant 24 heures. Le pH est ajusté à pH 8,8 et la solution est maintenue à 4 C° pendant encore 24 heures. Ce processus est répété jusqu'à ce que le pH soit ajusté à 8,0. Après 24 heures à pH 8,0, les protéines repliées peuvent être concentrées par ultrafiltration et appliquées à une colonne de filtration de gel en vue d'une purification.
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