EP0422124A1 - Liberation thermique d'une proteine recombinante dans un milieu de culture - Google Patents

Liberation thermique d'une proteine recombinante dans un milieu de culture

Info

Publication number
EP0422124A1
EP0422124A1 EP89908341A EP89908341A EP0422124A1 EP 0422124 A1 EP0422124 A1 EP 0422124A1 EP 89908341 A EP89908341 A EP 89908341A EP 89908341 A EP89908341 A EP 89908341A EP 0422124 A1 EP0422124 A1 EP 0422124A1
Authority
EP
European Patent Office
Prior art keywords
protein
host
nutrient medium
aqueous nutrient
substantially pure
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.)
Withdrawn
Application number
EP89908341A
Other languages
German (de)
English (en)
Other versions
EP0422124A4 (en
Inventor
Malcolm A. J. Finkelman
Timothy Kwok-Tim Lee
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.)
Genex Corp
Original Assignee
Genex Corp
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 Genex Corp filed Critical Genex Corp
Publication of EP0422124A1 publication Critical patent/EP0422124A1/fr
Publication of EP0422124A4 publication Critical patent/EP0422124A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates to methods for the isolation of substantially pure polypeptides from recombinant micro ⁇ organisms.
  • Proteins produced by host cells are normally trapped within the cells or secreted into the surrounding growth medium. In the former case, the cells must be ruptured to permit the desired protein to be isolated, whereas in the latter case, it can be separated from the growth media. Even in the case of secreted proteins, the preparation from which the protein is to be isolated is relatively complex, contain ⁇ ing a variety of other substances. Despite efficient separa ⁇ tion techniques, both the purity and the yield of the desired protein may be low. Lofdahl, S., et al ., PCT application, publication no. WO 84/03103, published 16 August 1984.
  • Lofdahl et al .. supra have developed methods for selectively isolating a desired protein or polypeptide by constructing a recombinant vector containing a DNA sequence coding for the desired protein or polypeptide which is operatively linked to a DNA sequence coding for protein A.
  • the expressed fusion protein is then selectively isolated by absorbing onto an IgG-supporting carrier, which binds protein A, followed by desorpt on of the fusion protein.
  • the fusion protein is then cleaved at a unique cleavage site with a cleavage agent, which may include proteases, hydroxylamine, cyanogen bromide or formic acid, to give the purified protein.
  • a cleavage agent which may include proteases, hydroxylamine, cyanogen bromide or formic acid
  • the invention relates to a method for the isolation of a substantially pure polypeptide expressed by a recombinant host comprising:
  • Figure 1 depicts a densitometric scan of a whole cell E coli extract of protein G separated by polyacrylamide electro- phoresis.
  • Figure 2 depicts a densitometric scan of the supernatant separated by polyacrylamide electrophoresis after heating an aqueous nutrient medium containing E. coli transformed with a vector containing a gene encoding protein G to 80 ⁇ C for 5 minutes.
  • the major peak is protein G.
  • the invention is directed toward a method for the isolation of substantially pure polypeptide from a recombinant bacterial host by culturing the host under protein-producing conditions, heating the aqueous nutrient medium to 50-100"C for a time not to exceed 1 hour, and isolating the substan ⁇ tially pure polypeptide so produced. Although longer heating times and higher temperatures may be utilized, they are not preferred since degradation of the protein occurs under these harsher conditions.
  • Preferred bacterial hosts include Gram-negative organ ⁇ isms, in particular, E. coli, Erwin a sp. and Klebsiella sp. The most preferred host is E. coli.
  • the aqueous nutrient medium is heated to 50- 100"C for about 5 minutes. Most preferably, the aqueous nutrient medium is heated to about 80°C for about 5 minutes. Under the most preferable conditions, substantially pure polypeptide is released into the culture media free from significant degradation.
  • the method has the added advantage that endogenous proteases are inactivated by the high tempera ⁇ tures of the method, thus preventing proteolysis of the polypeptide.
  • polypeptide is intended protein G or protein G variants having the i munoglobulin binding proper- ties of protein G, protein A or protein A variants having the immunoglobulin binding properties of protein A, and those polypeptides which may be produced in substantially pure form by the method of the invention, which include any peptide which does not irreversibly denature at 50-100 c C and which can penetrate the cell wall during the heating procedure.
  • proteins may include, but are not limited to, small proteins such as hormones, e.g., parathyroid hormone, growth hormone, ganadotropins (FSH, luteinizing hormone, chorionogonado- tropin), insulin, ACTH, prolactin, placenta! lactogen, melanocyte stimulating hormone, thyrotropin, calcitonin, enkephalin, angiotensin, and small cytokines.
  • hormones e.g., parathyroid hormone, growth hormone, ganadotropins (FSH, luteinizing hormone, chorion
  • the recombinant microorganism contains a vector containing the gene which encodes protein G.
  • Vectors which contain the genes which encode protein G and protein G variants which have the immunoglobulin binding properties of protein G are described, for example, in International Application PCT/US87/00329, co-pending U.S. Application Serial No. 063,959, filed June 19, 1987, and co- pending U.S. Application Serial No. 209,236, filed June 20, 1988, the disclosures of which are incorporated by reference herein in their entirety.
  • the vectors may incorporate promoters derived from, for example, bacteriophage, especially bacteriophage lambda, the E. coli tryptophan operon, the ______ coli lac operon, the E.
  • coli J-glucuronidase locus etc.
  • Suitable recombinant hosts include E. coli GX7820, J . coli GX7823, E. coli GX8464, and E. coli GX8465.
  • the production organism is the transformed host _____ coli GX1201 or E. coli GX6705, which contains the vector pGX5204 or a degenerate variant thereof.
  • polypeptides which are substantially one major band by SDS-PAGE polyacryla- mide electrophoresis and which contain only minor amounts of other proteins which normally contaminate a whole cell lysate, as evidenced by the presence of other minor bands.
  • the recombinant cells may be cultivated under any physiologically compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that support cell growth.
  • Recombinant protein-producing cultivation conditions will vary according to the type of vector used to transform the host cells.
  • certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals to the cell growth medium, to initiate the gene expression which results in the production of the recombinant polypeptide.
  • the term recombinant "protein-producing conditions,” as used herein, is not meant to be limited to any one set of cultivation conditions.
  • the expressed protein may be recovered from the fermenta ⁇ tion broth using any of the methods commonly known to those skilled in the art.
  • dead cells and insoluble debris are removed from the aqueous nutrient medium by filtration or centrifugation.
  • Protein G or other recombinant protein may then be purified from the fermentation media using standard procedures such as absorption to immobilized immuno ⁇ globulin, as described by Sjoquist, U.S. Patent No. 3,850,798 (1974), ion exchange or gel chromatography, precipitation (e.g., with ammonium sulfate), dialysis, filtration or a combination of these methods.
  • the method of the invention is not limited to expression of polypeptides which have leader sequences.
  • the invention does not depend on induction of filamentous growth of E. coli as described by Abrahamsen et al .. European Patent Application 0225860. Cell death is complete upon heating at 70-80°C; thus, induction of filamentous growth at these temperatures is not possible.
  • the fermenta ⁇ tion media is heated to 50-100°C in as little time as 5 minutes, there is little time for meaningful physiologically related morphological changes.
  • Soluble polypeptide for example protein G
  • Soluble polypeptide diffuses through the PG layer without significant impediment, by a dialysis-like mechanism.
  • the polypeptides which may be recovered according to the invention, as listed above, are those which remain soluble and do not denaturate upon heating.
  • Recombinant E. coli containing a vector which encodes a protein G variant having the immunoglobulin binding properties of protein G was cultivated in an aqueous nutrient medium (see Table 1) at 32 ⁇ C, 800 rpm, 1 vvm, pH 7.2 ⁇ 0.1 (titrants: 10% NaOH, 2M H3PO4). After cultivation at 32°C for 6-12 hours, expression of recombinant protein G was induced by raising the broth temperature to 42 ⁇ C for 1 hr. After 1 hr at 42°C, the broth temperature was reduced to 39 ⁇ C and maintained at that temperature for 3 hours.
  • heating at 80°C results in the release of substan ⁇ tially all of the protein G into the supernatant and retention of other proteins within the cells.
  • Example 1 The experiment in Example 1 was repeated using a 70°C heat treatment instead of an 80°C heat treatment to induce release of protein G. At this temperature, the release of protein G into the broth does not appear to be complete. Although substantially pure, the protein G appears to degrade after extended heating at 70°C as evidenced by fuzzy bands on the polyacrylamide electrophoresis gel.
  • Example 1 was repeated, except that the holding period at 80 ⁇ C was reduced to about 5 minutes. This brief exposure resulted in essentially quantitative release of substantially pure protein G as observed by SDS PAGE electrophoresis ( Figure 2). In addition, the apparent degradation of protein G due to prolonged heating (as evidenced by the widening of the protein G band on the gel) was greatly reduced.
  • Recombinant E. coli containing a vector which encodes Protein A strain NRRL 15910 (U.S. Patent No. 4,691,009) was cultivated in an aqueous medium (see Table 2) at 37°C, 800 rpm, 100 min., pH 7.2 ⁇ 0.1 (titrants: 10% NH 4 0H, 2 M H 3 P0 ). After growth leveled off, the broth was heated to 80 ⁇ C for 5 min, then cooled to 30 ⁇ C. SDS-PAGE analysis of the distribu ⁇ tion of recombinant Protein A demonstrated that prior to heating, the Protein A was present in the cells and very little was detectable in the supernatant.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention se rapporte à un procédé pour l'isolation d'un polypeptide pratiquement pur contenu dans un hôte bactérien recombinant, par la culture dudit hôte dans des conditions de production d'une protéine recombinante, suivie du chauffage du milieu nutritif aqueux à une température de 50-100°C pendant une durée n'excédant pas une heure, puis de la récupération du polypeptide quasiment pur ainsi produit.
EP19890908341 1988-06-27 1989-05-05 Thermal release of recombinant protein into culture media Withdrawn EP0422124A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21190488A 1988-06-27 1988-06-27
US211904 1988-06-27

Publications (2)

Publication Number Publication Date
EP0422124A1 true EP0422124A1 (fr) 1991-04-17
EP0422124A4 EP0422124A4 (en) 1991-10-16

Family

ID=22788768

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19890908341 Withdrawn EP0422124A4 (en) 1988-06-27 1989-05-05 Thermal release of recombinant protein into culture media

Country Status (3)

Country Link
EP (1) EP0422124A4 (fr)
JP (1) JPH03505401A (fr)
WO (1) WO1990000200A1 (fr)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0600247A3 (fr) * 1992-11-05 1995-05-10 Hoechst Ag Méthode de désactivation sélective de protéine non désirable dans un mélange, protéinique, par les radiations d'un four à microondes.
US5985304A (en) * 1998-02-25 1999-11-16 Battelle Memorial Institute Barrier preventing wood pest access to wooden structures
US6596509B1 (en) 1998-07-10 2003-07-22 Cornell Research Foundation, Inc. Recombinant constructs and systems for secretion of proteins via type III secretion systems
AU2002316811A1 (en) 2001-06-28 2003-03-03 Novo Nordisk A/S Stable formulation of modified glp-1
EP2823809B1 (fr) 2002-06-28 2016-11-02 Protiva Biotherapeutics Inc. Procédé et appareil pour la production de liposomes
WO2004048588A1 (fr) * 2002-11-26 2004-06-10 Novo Nordisk A/S Procede pour purifier un produit de fermentation
JP2007524592A (ja) 2003-06-03 2007-08-30 ノボ・ノルデイスク・エー／エス 安定化された薬学的ペプチド組成物
ATE541582T1 (de) 2003-06-03 2012-02-15 Novo Nordisk As Stabilisierte pharmazeutische glp-1 peptid zusammensetzungen
EP2394656B1 (fr) 2003-11-20 2023-12-13 Novo Nordisk A/S Formulations pharmaceutiques comprenant le peptide liraglutide et le propylene glycol
PL1789434T3 (pl) 2004-08-31 2014-07-31 Novo Nordisk As Zastosowanie tris(hydroksymetylo)aminometanu do stabilizacji peptydów, polipeptydów i białek
CA2586771A1 (fr) 2004-11-12 2006-05-18 Novo Nordisk A/S Formulations stables de peptides
US20100233783A1 (en) * 2005-03-24 2010-09-16 Straumann Holding Ag Novel method for protein purification
RU2646103C2 (ru) * 2016-03-30 2018-03-01 Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) Способ выделения ренатурированного белка g из маркированного полиакриламидного геля
TWI762706B (zh) 2017-08-24 2022-05-01 丹麥商諾佛 儂迪克股份有限公司 Glp-1組成物及其用途
US20230093542A1 (en) 2020-02-18 2023-03-23 Novo Nordisk A/S Glp-1 compositions and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0149514A2 (fr) * 1984-01-12 1985-07-24 Simon-Hartley Limited Agent floculant
EP0225860A2 (fr) * 1985-12-13 1987-06-16 Kabi Pharmacia AB Méthode pour transmettre des produits de gènes à un milieu de croissance de bactéries gram-négatives
EP0234863A2 (fr) * 1986-02-22 1987-09-02 Bovril Limited Procédé de désintégration de cellules

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1187512A (en) * 1966-08-05 1970-04-08 Kyowa Hakko Kogyo Kk Process for Extracting Proteins from Micro-Organisms

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0149514A2 (fr) * 1984-01-12 1985-07-24 Simon-Hartley Limited Agent floculant
EP0225860A2 (fr) * 1985-12-13 1987-06-16 Kabi Pharmacia AB Méthode pour transmettre des produits de gènes à un milieu de croissance de bactéries gram-négatives
EP0234863A2 (fr) * 1986-02-22 1987-09-02 Bovril Limited Procédé de désintégration de cellules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9000200A1 *

Also Published As

Publication number Publication date
WO1990000200A1 (fr) 1990-01-11
EP0422124A4 (en) 1991-10-16
JPH03505401A (ja) 1991-11-28

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