EP0804461A1 - Procede de repliement de proteines telles que l'hirudine ou le facteur de croissance epidermique de recombinaison - Google Patents

Procede de repliement de proteines telles que l'hirudine ou le facteur de croissance epidermique de recombinaison

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Publication number
EP0804461A1
EP0804461A1 EP95926859A EP95926859A EP0804461A1 EP 0804461 A1 EP0804461 A1 EP 0804461A1 EP 95926859 A EP95926859 A EP 95926859A EP 95926859 A EP95926859 A EP 95926859A EP 0804461 A1 EP0804461 A1 EP 0804461A1
Authority
EP
European Patent Office
Prior art keywords
protein
process according
proteins
hirudin
concentration
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Withdrawn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
EP95926859A
Other languages
German (de)
English (en)
Inventor
Jui Yoa Chang
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.)
Novartis AG
Original Assignee
Ciba Geigy AG
Novartis 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, Novartis AG filed Critical Ciba Geigy AG
Priority to EP95926859A priority Critical patent/EP0804461A1/fr
Publication of EP0804461A1 publication Critical patent/EP0804461A1/fr
Withdrawn legal-status Critical Current

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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)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé permettant de préparer une protéine repliée correctement, biologiquement active, en présence d'un agent dénaturant tel que l'urée ou l'hydrochlorure de guanidine, et de séparer la protéine correctement repliée directement de celui-ci. Ce procédé peut être mis en ÷uvre, par exemple, lors de la renaturation de protéines de recombinaison telles que l'hirudine ou le facteur de croissance épidermique.
EP95926859A 1994-07-25 1995-07-12 Procede de repliement de proteines telles que l'hirudine ou le facteur de croissance epidermique de recombinaison Withdrawn EP0804461A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP95926859A EP0804461A1 (fr) 1994-07-25 1995-07-12 Procede de repliement de proteines telles que l'hirudine ou le facteur de croissance epidermique de recombinaison

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP94810437 1994-07-25
EP94810437 1994-07-25
EP95926859A EP0804461A1 (fr) 1994-07-25 1995-07-12 Procede de repliement de proteines telles que l'hirudine ou le facteur de croissance epidermique de recombinaison
PCT/EP1995/002720 WO1996003425A1 (fr) 1994-07-25 1995-07-12 Procede de repliement de proteines telles que l'hirudine ou le facteur de croissance epidermique de recombinaison

Publications (1)

Publication Number Publication Date
EP0804461A1 true EP0804461A1 (fr) 1997-11-05

Family

ID=8218291

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95926859A Withdrawn EP0804461A1 (fr) 1994-07-25 1995-07-12 Procede de repliement de proteines telles que l'hirudine ou le facteur de croissance epidermique de recombinaison

Country Status (9)

Country Link
EP (1) EP0804461A1 (fr)
JP (1) JPH11505507A (fr)
AU (1) AU3109795A (fr)
CA (1) CA2194177A1 (fr)
FI (1) FI970230A7 (fr)
IL (1) IL114699A0 (fr)
NO (1) NO970081D0 (fr)
WO (1) WO1996003425A1 (fr)
ZA (1) ZA956137B (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3734634B2 (ja) 1999-03-03 2006-01-11 ヒゲタ醤油株式会社 タンパク質の活性化方法及び該装置
US7521479B2 (en) 2001-04-16 2009-04-21 Panacea Pharmaceuticals, Inc. Methods of treating prion disease in mammals
WO2003097669A2 (fr) * 2002-05-17 2003-11-27 Københavns Universitet Procede de purification de proteines denaturees presentant une configuration de liaisons de disulfide voulue

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR79124B (fr) * 1982-12-22 1984-10-02 Genentech Inc
ES8800957A1 (es) * 1985-02-22 1987-12-01 Monsanto Co Un metodo para la solubilizacion y renaturalizacion de proteina somatotropina
GB8927546D0 (en) * 1989-12-06 1990-02-07 Ciba Geigy Process for the production of biologically active tgf-beta
JP3200850B2 (ja) * 1991-02-26 2001-08-20 味の素株式会社 ヒトｂｃｄｆの精製法
US5290920A (en) * 1992-04-16 1994-03-01 Allelix Biopharmaceuticals Inc. Method of purifying human epidermal growth factor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9603425A1 *

Also Published As

Publication number Publication date
JPH11505507A (ja) 1999-05-21
CA2194177A1 (fr) 1996-02-08
NO970081L (no) 1997-01-09
NO970081D0 (no) 1997-01-09
IL114699A0 (en) 1995-11-27
AU3109795A (en) 1996-02-22
FI970230A0 (fi) 1997-01-20
WO1996003425A1 (fr) 1996-02-08
ZA956137B (en) 1996-03-07
FI970230L (fi) 1997-01-20
FI970230A7 (fi) 1997-01-20

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