WO1996003425A1 - Process for folding of proteins like recombinant hirudin or epidermal growth factor - Google Patents

Process for folding of proteins like recombinant hirudin or epidermal growth factor Download PDF

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Publication number
WO1996003425A1
WO1996003425A1 PCT/EP1995/002720 EP9502720W WO9603425A1 WO 1996003425 A1 WO1996003425 A1 WO 1996003425A1 EP 9502720 W EP9502720 W EP 9502720W WO 9603425 A1 WO9603425 A1 WO 9603425A1
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WIPO (PCT)
Prior art keywords
protein
process according
proteins
hirudin
concentration
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Application number
PCT/EP1995/002720
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English (en)
French (fr)
Inventor
Jui Yoa Chang
Original Assignee
Ciba-Geigy Ag
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 Ciba-Geigy Ag filed Critical Ciba-Geigy Ag
Priority to JP8505402A priority Critical patent/JPH11505507A/ja
Priority to EP95926859A priority patent/EP0804461A1/en
Priority to MX9700658A priority patent/MX9700658A/es
Priority to AU31097/95A priority patent/AU3109795A/en
Publication of WO1996003425A1 publication Critical patent/WO1996003425A1/en
Priority to NO970081A priority patent/NO970081D0/no
Priority to FI970230A priority patent/FI970230A/fi

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Classifications

    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a process for the preparation of a biologically active and correctly folded protein in the presence of a denaturing agent.
  • CSF-1 colony stimulating factor-1
  • the procedures described involve the steps of initial solubilization of CSF-1 monomers isolated from inclusion bodies under reducing conditions in a chaotropic environment comprising urea or guanidine hydrochloride, refolding which is achieved by stepwise dilution of the chaotropic agents, and final oxidation of the refolded molecule in the presence of a redox-system.
  • IL-2 interieukin-2
  • IL-2 isolated from retractile bodies is denatured under reducing conditions with 6M guanidine hydrochloride, the soluble IL-2 is oxidized by a controlled oxidation in the presence of Cu 2+ ions, and the oxidized IL-2 refolded by reducing the concentration of the denaturant in the solution.
  • Interieukin-2 and interferon- ⁇ have been refolded using SDS for solubilization and Cu 2+ ions as oxidation promoters of the fully reduced protein (US-A-4572798).
  • the process for isolating recombinant retractile proteins as described in US-A-4620948 involves strong denaturing agents to solubilize the proteins, reducing conditions to facilitate correct folding and denaturant replacement in presence of air or other oxidizing agents to reform the disulfide bonds.
  • a method for renaturing unfolded proteins including cytochrome c, ovalbumin and trypsin inhibitor by reversibly binding of the denatured protein to a solid matrix and stepwise renaturing it by diluting the denaturant is disclosed in WO 86/5809.
  • the foregoing references are merely representative of a huge amount of literature dealing with the refolding of non-native proteins derived from different sources.
  • Guanidine hydrochloride (GdmCI) and urea are the best known denaturants for unfolding and inactivating proteins. Although the mechanism of their actions remains to be fully understood, it is generally evident that they disrupt the non-covalent interactions which stabilize the native conformation. The detrimental effect of GdmCI and urea has also been illustrated during the folding of proteins, which usually leads to the formation of inactive, scrambled species (Haber and Anfinsen, J. Biol. Chem. (1962), 237, 1839-1844; Weissman and Kim Science (1991 ), 253, 1386-1393). On the other hand, denaturants are potent agents for solubilizing intractable proteins, such as immunoglobulins and membrane components etc.
  • recombinant proteins expressed in an Escherichia coli system often face this problem of protein solubility. These proteins are frequently found in insoluble inclusion bodies and require solubilization by strong denaturant as the essential step to refold and generate the active conformation.
  • the current invention provides a process for the production of a correct folded protein or a salt thereof, characterized in that the protein is treated with a buffer comprising a denaturant, and the correct folded protein is separated therefrom directly, wherein the denaturant is selected from the group consisting of guanidine hydrochloride in a concentration from 3 to 7 M and urea in a concentration from 6 to 10 M.
  • the wording correct folded protein stands for a protein that is in the native conformation and/or shows a biological activity like the enzymatic activity or the binding property of the native protein.
  • the inventive process is applicable to any protein or protein fragment that has to be folded into a correct folded conformation and that establishes an equilibrium between a not correct and a correct folded conformation in presence of the denaturant. This is usually the case for proteins that are not irreversibly denatured by the denaturant.
  • the ability of a protein to establish said equilibrium can easily be monitored by standard methods that provide information on the folding of proteins in solution like NMR or circular dicroism.
  • the protein to be refolded by the inventive process may be from almost any source, a special pretreatment is not necessary but not excluded.
  • a recombinant protein that is stored in the producing host in form of inclusion bodies can be refolded by simply separating the inclusion bodies from the rest of the cell debris, solubilizing the proteins of the inclusion bodies with denaturant and isolating the correct folded protein therefrom.
  • the protein is not stored in form of inclusion bodies it is possible to enrich the protein only to some extend using, e.g., a precipitation step, solubilize the protein with the denaturant and isolate the pure and correct folded protein therefrom.
  • telomeres For recombinant proteins or natural proteins that are not in a correct folded conformation after isolation it is possible to solubilize these protein after isolation in the denaturant and isolate the correct folded fraction therefrom.
  • suitable proteins are hirudin, epidermal growth factor, potato carboxypeptidase inhibitor (PCI) and bovine pancreatic trypsin inhibitor (BPTI), IGF-1 , C5a- antagonist, TGF- ⁇ .
  • hirudin as used in this invention is intended to embrace all desulfatohirudin compounds described in literature or obtainable from a transformed microorganism strain containing DNA which codes for a desulfatohirudin or a derivative thereof.
  • Such hirudins are, for example, desulfatohirudin derivatives HV1 , HV2 and HV3 (PA), as well as other hirudin proteins as described e.g. by M. Scharf et al. (FEBS Lett., 255 (1989), 105-110) and EP-A-347376.
  • hirudin derivatives or shorter fragments having hirudin activity are also covered by the term "hirudin”.
  • Such fragments and derivatives are, for example, C-terminally shortened desulfatohirudins.
  • a and B represent percentages of native protein and scrambled species presented in equilibrium. A and B, both are smaller than 1 , can be readily derived from the equilibrium constant.
  • Another or additional possibility to shift the equilibrium towards the correct folded conformation is the addition of substances that promote correct folding like metal salts that stabilize the correct conformation or removing the native species using solid bound ligand that binds specifically to the native structure.
  • the correct folded and the not correct folded protein are separated continuously or discontinuously.
  • the concentration of guanidine hydrochloride is preferably from 4 to 6 M and the concentration of urea is preferably 7 to 9 M.
  • the process is carried out in presence of a reducing agent with a redoxpotential from -0.20 to -0.30 such as glutathione, cysteine or ⁇ -mercaptoethanol which is preferred.
  • concentration of the reducing agent is preferably from 0.05 to 1 mM and more preferably from 0.1 to 0.5 mM.
  • the denatu ration buffer or the buffer to that the correct folded protein is isolated may also contain additional compound that promote folding or prevent undesired side reactions.
  • additional compound that promote folding or prevent undesired side reactions.
  • further denaturants like SDS and Triton or metal ions, further reducing agents, oxidizing agents, complexing agents like EDTA or co-enzymes.
  • the correct folded protein may be separated from the not corrected folded protein by any process that is able to distinguish said two forms.
  • Said processes are, e.g., based on a difference in mobility, shape, reactivity or binding properties. Examples for suitable processes are antibody based, membrane based, electrophoretic or chromatographic separations like gel electrophoresis, gel filtration, thin layer chromatography (TLC), HPLC, affinity chromatography or separation via a selective membrane.
  • TLC thin layer chromatography
  • HPLC highLC
  • affinity chromatography affinity chromatography
  • the folding reactions is carried out preferably at a temperature that promotes the establishment of an equilibrium and does not irreversibly denature the protein. Therefore, the applied temperature mainly depends on the stability of the protein and the separation procedure for the correct folded protein. For example, certain proteins of thermophile microorganisms are stable at 60°C and above while proteins that originate from not thermophilic microorganisms might sometimes enter irreversible modifications at 40°C or below.
  • Fig. 1 is a HPLC protocol of the hirudin core domain (Hir 1" 9 ) in presence of 5 M guanidine hydrochloride and 0.25 mM ⁇ -mercaptoethanol.
  • Fig. 2 is a HPLC protocol of epidermal growth factor in presence of 3 M guanidine hydrochloride and 0.25 mM ⁇ -mercaptoethanol.
  • Example 1 Production and isolation of the hirudin core domain (Hir )
  • Recombinant desulfated hirudin was is isolated from Saccharomyces cerevisiae as described in Meyhack et al. (Thromb. Res. Suppl. VII (1987), 33).
  • the isolated desulfatohirudin is dissolved in 50 mM ammoniumbicarbonate buffer pH 8.0 at a concentration of 5 mg/ml and digested with chymotrypsin (0.25 mg/ml) at room temperature for 16 h. The digestion is terminated by addition of trif luoroacetic acid to a final concentration of 0.8 % and the core domain (Hir 1"49 ) is isolated by HPLC.
  • Solvent A 0.1 % trif luoroacetic acid in water
  • Solvent B 0.1 % trif luoroacetic acid in acetonitrile
  • the starting material for the folding experiments fully reduced / denatured core domain of hirudin [R], is prepared by the following method:
  • the hirudin core domain from examplel (2 mg/ml) is dissolved in Tris-HCI buffer (0.5 M, pH 8.5) containing 5 M of guanidine chloride (GdmCI) and 30 mM of dithiothreitol. Reduction and denaturation are carried out at 23°C for 90 min.
  • the sample is passed through a PD-IO* column (Pharmacia) equilibrated in 0.1 M Tris-HCI buffer (pH 8.5). Desalting takes about 1-2 min and the sample is immediately used in the folding experiments.
  • Example 3 Folding of hirudin in the presence of guanidine hydrochloride
  • the samples are diluted to a final protein concentration of 1 mg/ml; containing 0.1 M Tris- HCI buffer (pH 8.5), 5 M guanidine hydrochloride and 0.25 mM ⁇ -mercaptoethanol. After 24 h incubation at room temperature, the native hirudin is separated from scrambled hirudin via HPLC.
  • Solvent A 0.1 % trif luoroacetic acid in water
  • Solvent B 0.1 % trif luoroacetic acid in acetonitrile
  • HPLC-protocol is given in Figure 1.
  • the amount of native hirudin is calculated to 60% ⁇ 5 % and the K ⁇ to 0.67 ⁇ 0.15.
  • the traction containing the scrambled hirudin is lyophilized and dissolved in 0.1 M Tris-HCI buffer (pH 8.5) containing 5 M guanidine hydrochloride and 0.25 mM ⁇ -mercaptoethanol to a final protein concentration of 1 mg/ml. After 24 h incubation, the native hirudin is separated from scrambled hirudin via HPLC as described above Lyophilization and renaturation is carried out for a third time as described above.
  • the activity of the recovered hirudin is proved by the ability to inhibit human ⁇ -thrombin from digesting Chromozym (Boehringer Mannheim).
  • the reaction is carried at 22°C in 67 mM Tris-HCI buffer (pH 8.0) containing 133 mM NaCI and 0.13 % polyethylene glycol 6000.
  • the rate of digestion was followed at 405 nm for a period of 2 min.
  • the concentration of substrate is 200 mM.
  • the concentration of thrombin is adjusted in between 2.5 and 25 nM.
  • the recovered protein is carboxymethylated with 0.2 M iodoacetic acid in Tris-HCI buffer (0.5 M, pH 8.5) for 30 min and desalted through a PD-10 ® column equilibrated with ammonium bicarbonate solution (50 mM, pH 8.0).
  • the disulfide contents is determined by amino acid analysis (Chang and Knecht, Anal. Biochem. (1991), 197, 52-58) and mass spectrometry (Chatrenet and Chang, J. Biol. Chem. (1992), 267, 3038-3043).
  • the renaturation is carried out as described in example 3 with the sole difference that 8 M urea is used instead of 5 M guanidine hydrochloride.
  • the amount of native hirudin is calculated to 90 % ⁇ 5 % and the K ⁇ q to 0.11 ⁇ 0.06.
  • Epidermal growth factor is provided by Protein Institute Inc. (Broomall, USA) and is denatured as described for hirudin in example 2.
  • the renaturation is carried out as described in example 3 with the sole difference that EGF is used instead of hirudin and 3 M guanidine hydrochloride is used instead of 5 M guanidine hydrochloride.
  • the HPLC-protocol is given in Figure 2.
  • the disulfide content is determined as described in example3.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/EP1995/002720 1994-07-25 1995-07-12 Process for folding of proteins like recombinant hirudin or epidermal growth factor WO1996003425A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP8505402A JPH11505507A (ja) 1994-07-25 1995-07-12 組換えヒルジンまたは表皮増殖因子のようなタンパク質の折りたたみ方法
EP95926859A EP0804461A1 (en) 1994-07-25 1995-07-12 Process for folding of proteins like recombinant hirudin or epidermal growth factor
MX9700658A MX9700658A (es) 1995-07-12 1995-07-12 Proceso para el plegamiento de proteinas como hirudina recombinante o factor de crecimiento epidermico.
AU31097/95A AU3109795A (en) 1994-07-25 1995-07-12 Process for folding of proteins like recombinant hirudin or epidermal growth factor
NO970081A NO970081D0 (no) 1994-07-25 1997-01-09 Fremgangsmåte for folding av proteiner som rekombinant hirudin eller epidermisk vekstfaktor
FI970230A FI970230A (fi) 1994-07-25 1997-01-20 Menetelmä proteiinien, kuten yhdistelmä-DNA-hirudiinin tai -epidermaalisen kasvutekijän laskostamiseksi

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP94810437.7 1994-07-25
EP94810437 1994-07-25

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WO1996003425A1 true WO1996003425A1 (en) 1996-02-08

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EP (1) EP0804461A1 (no)
JP (1) JPH11505507A (no)
AU (1) AU3109795A (no)
CA (1) CA2194177A1 (no)
FI (1) FI970230A (no)
IL (1) IL114699A0 (no)
NO (1) NO970081D0 (no)
WO (1) WO1996003425A1 (no)
ZA (1) ZA956137B (no)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1035129A1 (en) * 1999-03-03 2000-09-13 Higeta Shoyu Co., Ltd. A method of activating protein and an apparatus therefor
WO2003097669A2 (en) * 2002-05-17 2003-11-27 Københavns Universitet Method for purifying denatured proteins having a desired disulfide bond configuration
US7521479B2 (en) 2001-04-16 2009-04-21 Panacea Pharmaceuticals, Inc. Methods of treating prion disease in mammals

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0114506A1 (en) * 1982-12-22 1984-08-01 Genentech, Inc. Methods of purification and reactivation of precipitated heterologous proteins
EP0192629A2 (en) * 1985-02-22 1986-08-27 Monsanto Company Method of somatotropin solubilization and naturation
EP0433225A1 (en) * 1989-12-06 1991-06-19 Ciba-Geigy Ag Process for the production of biologically active protein (e.g. TGF)
EP0529086A1 (en) * 1991-02-26 1993-03-03 Ajinomoto Co., Inc. Processes for purifying human bcdf
WO1993021224A1 (en) * 1992-04-16 1993-10-28 Allelix Biopharmaceuticals Inc. Ultrapure human epidermal growth factor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0114506A1 (en) * 1982-12-22 1984-08-01 Genentech, Inc. Methods of purification and reactivation of precipitated heterologous proteins
EP0192629A2 (en) * 1985-02-22 1986-08-27 Monsanto Company Method of somatotropin solubilization and naturation
EP0433225A1 (en) * 1989-12-06 1991-06-19 Ciba-Geigy Ag Process for the production of biologically active protein (e.g. TGF)
EP0529086A1 (en) * 1991-02-26 1993-03-03 Ajinomoto Co., Inc. Processes for purifying human bcdf
WO1993021224A1 (en) * 1992-04-16 1993-10-28 Allelix Biopharmaceuticals Inc. Ultrapure human epidermal growth factor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
B. CHATRENET AND J.-Y. CHANG: "The Folding of Hirudin Adopts a Mechanism of Trial and Error", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 5, 15 February 1992 (1992-02-15), BALTIMORE, MD US, pages 3038 - 3043 *
L.O. NARHI ET AL.: "Circular dichroism of reduced and oxidized recombinant human epidermal growth factor", INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH, vol. 39, no. 2, COPENHAGEN DK, pages 182 - 187 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1035129A1 (en) * 1999-03-03 2000-09-13 Higeta Shoyu Co., Ltd. A method of activating protein and an apparatus therefor
US6479257B1 (en) 1999-03-03 2002-11-12 Higeta Shoyu Co., Ltd. Method of activating protein and an apparatus therefor
US7521479B2 (en) 2001-04-16 2009-04-21 Panacea Pharmaceuticals, Inc. Methods of treating prion disease in mammals
WO2003097669A2 (en) * 2002-05-17 2003-11-27 Københavns Universitet Method for purifying denatured proteins having a desired disulfide bond configuration
WO2003097669A3 (en) * 2002-05-17 2004-03-18 Univ Koebenhavn Method for purifying denatured proteins having a desired disulfide bond configuration

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Publication number Publication date
IL114699A0 (en) 1995-11-27
FI970230A0 (fi) 1997-01-20
FI970230A (fi) 1997-01-20
NO970081L (no) 1997-01-09
AU3109795A (en) 1996-02-22
JPH11505507A (ja) 1999-05-21
EP0804461A1 (en) 1997-11-05
ZA956137B (en) 1996-03-07
NO970081D0 (no) 1997-01-09
CA2194177A1 (en) 1996-02-08

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