JPH11505507A - Methods for folding proteins such as recombinant hirudin or epidermal growth factor - Google Patents

Abstract

  (57) [Summary] The present invention relates to the preparation of biologically active, correctly folded proteins in the presence of denaturing agents, such as urea or guanidine hydrochloride, and the separation of directly folded proteins therefrom. This method can be applied, for example, to the regeneration of recombinant proteins such as hirudin or epidermal growth factor.

Description

DETAILED DESCRIPTION OF THE INVENTION Methods for folding proteins such as recombinant hirudin or epidermal growth factor   The present invention relates to a biologically active and correctly folded tamper in the presence of a denaturing agent. The present invention relates to a method for preparing a substance.   The correct folding of genetically engineered proteins is a major part of protein science. It is a problem. The problem is, for example, in the case of disulfide-containing proteins, especially Serious, often requires denaturation and refolding to become active . Disulfide formation is a case of post-translational modification. It folds protein And usually occurs faithfully in vivo. However, in vitro regeneration experiments Then, many proteins are recovered only a little and some are completely native Does not fold into the conformation of. To overcome this problem, a number of compounds, For example, a mixture of reduced / oxidized glutathione (Creighton "Methods Enzymol."131   83-106, 1986) and protein disulfide isomerase (PDI) (Morin, J. .E. And Dixon, J.E. "Methods Enzymol."113  541-547, 1985) It has been used routinely to promote disulfide formation. But its wide Despite ubiquitous use, the right mechanism for those features is And its application has been done in a trial and error manner.   Individual proteins produced in bacterial hosts, or otherwise denatured Or report a refolding attempt on a non-native protein Numerous publications have been published. After expression in E. coli, the biologically active Ronnie stimulating factor-1 (CSF- The formation of 1) was described in WO 88/8003 and by Halenbeck et al. "Biotechnology"7  710-715 (1989). Stated Reduced procedure in a chaotropic environment containing urea or guanidine The first solubilization step of CSF-1 monomer isolated from inclusion bodies below, chaotropic The refolding stage achieved by the stepwise dilution of the agent and the presence of a redox system Including the last oxidation step of the refolded molecule in International Publication No. 88/8849 In the description, 6M of interleukin-2 (IL-2) isolated from a refractive body was used. Guanidine hydrochloride is modified under certain reducing conditions to convert its soluble IL-2 to Cu²⁺ion Oxidation by the presence of controlled oxidation reduces the concentration of denaturants in solution Thereby refolding the oxidized IL-2. A method for collecting interleukin-2 (IL-2) is described. Interroy Kin-2 and interferon-β are SDS for solubilization and completely reduced Cu as a pro-oxidant for oxidized proteins²⁺Refolding by using ions (US Pat. No. 4,572,798). As described in U.S. Pat.No. 4,620,948 , A method for isolating a recombinant refractive protein, for dissolving the protein Strong denaturants, reducing conditions and disulfide bonds to promote correct folding The presence of denaturing agents in the presence of air or other oxidizing agents for the reformation of Solid Reversible binding of the denatured protein to the matrix and dilution of the denaturant Cytochrome C, ovalbumin and trypsin inhibitor A method for the regeneration of unfolded proteins, including, No. 86/5809. The preceding references have been derived from various sources. Only represent a vast literature dealing with non-intrinsic protein refolding Absent. On the other hand, those skilled in the art Know that success cannot be predicted. Unsuccessful experiments usually reported Not. Any of the reported folding conditions resulted in several cysteine residues, Therefore, a large number of intramolecular disulfide bonds, which are necessary for activity However, the assurance that a given denatured protein containing Not necessarily.   Guanidine hydrochloride (GdmCl) and urea unfold and inactivate proteins Are best known as denaturants. Are their mechanisms of action completely understood? However, they break the non-covalent reactions that stabilize the original higher-order structure. Breaking is usually obvious. The detrimental effects of GdmCl and urea are Although described during convolution, it is usually inert and confusing (scrambl ed) leads to the formation of species (Harber and Anfinsen "J. Biol. Chem."273, No. 1839-18 44 (1962); Weissman and Kim "Science"253, Pp. 1386-1393 (1991)) . On the other hand, denaturants are difficult to process proteins such as immunoglobulins and membrane components It is a powerful drug for dissolving etc. For example, a set expressed in an E. coli strain Recombinant proteins often face this problem of protein solubility. These tampa Quenching is often found in insoluble inclusion bodies and refolds to form active conformations. As an essential step, dissolution requires the use of strong denaturing agents.   Surprisingly, correctly folded in the presence of denaturing agents and equilibrium conditions It has now been discovered that proteins are produced and can be directly isolated therefrom. The finding is that recombinant proteins are dissolved using denaturing agents and the It can be isolated in its original, properly folded, conformation without removing the active agent. It greatly facilitates the preparation of these proteins. The presence of higher concentrations of denaturants is Inactivates normal protease and eliminates the need for protease inhibitors To   Thus, the present invention produces a correctly folded protein and its salts Treating the protein with a buffer containing a denaturing agent, Directly separates poorly folded proteins, the denaturant of which has a concentration of 3-7M Being selected from the group consisting of guanidine hydrochloride and urea at a concentration of 6-10M. Providing the above method.   The term correctly folded protein is its native conformation And / or biological activities such as the enzymatic activity or binding properties of its native protein Represents a protein that shows sex.   The method of the present invention must be folded into a correctly folded higher order structure. Conformation and conformation not correctly folded due to the presence of denaturants Any protein or protein that establishes an equilibrium with a tightly folded conformation Or to protein fragments. This is usually irreversibly denatured by denaturing agents This is the case for proteins that are not performed. The ability of the protein to establish the equilibrium Force can be used to fold proteins in solution, such as NMR or circular dichroism It can be easily monitored by standard methods that provide information about   Proteins folded by the method of the present invention can be derived from almost any source. And does not require special pre-treatment, but excludes it. There is no. For example, recombinant proteins stored in inclusion bodies in the producing host Simply separate the inclusion bodies from the rest of the cell debris and remove the correctly folded By isolating the protein, it can be refolded. protein If is not stored in the form of inclusion bodies, the tamper Simply concentrate to a certain extent and use a denaturant to Protein from which the pure, correctly folded protein is isolated It is possible to After isolation, recombination is not a correctly folded conformation, recombination For proteins or native proteins, after isolation in denaturing Lysed protein from which correctly folded fractions can be isolated Noh.   Examples of suitable proteins are hirudin, epidermal growth factor, potato carboxypope Peptidase inhibitor (PCl) and bovine pancreatic trypsin inhibitor (BPTI), ie, IGF -1, C5a-antagonist, TGF-β. A particularly preferred protein is leech Gin and epidermal growth factor (EGF).   The term "hirudin" as used in the present invention is defined as any of the terms described in the literature. Contains DNA encoding desulfatohirudin or desulfatohirudin All desulfatohirudin available from the transformed microorganism. Or derivatives thereof. The hirudin, for example, Rufatohirudin derivatives HV1, HV2 and HV3 (PA) and M. M. Scharf and others ("FEBS Lett."255Pp. 105-110 (1989)) and Europe Another hirudin protein described in State Patent Publication No. 347376. The term hirudin derivative or short fragment of hirudin having hirudin activity is also referred to as "hiruji It is understood that it is included in ". Such fragments and derivatives are, for example, C-terminal Desulfato hirudin shortened at the end.   Under denaturants, species that are not correctly folded (confused) during equilibrium Because there are unfolded (original) proteins, the original higher-order structure The condition may be designed so as to select and tilt the balance. One possible approach is The removal of the original seed continuously throughout the folding. Other methods are simply mixed Re-disturbed seed Is to circulate. This is to isolate the confused species and give rise to the original species By re-equilibrating them in a denaturing agent. Selected variables Knowing the equilibrium constant under sexual conditions, the total yield of the original protein is calculated using the following equation can do.         Yield = A. (1 + B + B)^Two + ... + Bⁿ ) Where n represents the number of recycles and A and B are the native proteins present in equilibrium. And% of species confused. A and B (both smaller than 1) are easy from equilibrium constants Can be obtained.   Move the equilibrium towards the correct folding conformation, other or additional Possibilities encourage correct folding, such as metal salts that stabilize the correct conformation. The addition of a substance that proceeds or the ligand bound to the solid that specifically binds to the original structure The removal of the original seed used.   In a preferred embodiment of the invention, correctly folded proteins and correctly folded Unfolded proteins are separated continuously or discontinuously.   In the method of the present invention, correctly folded To establish an equilibrium between unfolded and correctly folded conformations. Any denaturing agent that allows it can be used.   The concentration of guanidine hydrochloride is preferably 4-6M and the concentration of urea is preferably Is 7-9M.   Proteins that fold correctly with one or more disulfide bonds If there is confusion in the folded conformation, add a reducing agent or redock I recommend a system.   Advantageously, a reducing agent having a redox potential of -0.20 to 0.30, such as glue Tathione, cysteine or β-mercaptoethanol (Preferred). The concentration of the reducing agent is preferably 0.05 to The concentration is 1 mM, more preferably 0.1 to 0.5 mM.   Denaturant buffers or buffers from which correctly folded proteins are isolated It may also contain additional compounds that promote folding or prevent unwanted side reactions .   Examples include further denaturation such as SDS, Triton or metal ions Agents, further reducing agents, oxidizing agents, complexing agents such as EDTA or coenzymes.   A correctly folded protein is a protein that is not correctly folded From which the two forms can be distinguished and separated by any method You. The method is based, for example, on mobility, shape, reactivity or binding properties. Suitable Examples of intelligent methods include gel electrophoresis, gel filtration, thin layer chromatography (TLC), HPL C, antibodies, such as affinity chromatography or selective membrane separation -Based separation, membrane-based separation, electrophoretic separation or chromatography It is separation. In a preferred embodiment of the invention, the correctly folded protein is HP Separated by LC, TLC or affinity chromatography.   Discontinuous separation of correctly folded proteins, such as residual Concentrate or dilute the solution to re-fit the conditions needed to establish the equilibrium I have to do it. For example, correctly folded protein and correct The folded protein is isolated separately, for example using an HPLC column, and Concentrate the fraction containing the unfolded protein and concentrate it under denaturing conditions. It is possible to dissolve again below. If the separation process is performed continuously, The concentration of essential components is monitored, for example by spectroscopic means, and is continuously adapted be able to.   The folding reaction preferably promotes the establishment of equilibrium and irreversibly converts the protein. It is performed at a temperature that does not denature. Therefore, the applied temperature mainly depends on the protein Depends on quality stability and separation procedures for correctly folded proteins . For example, certain proteins of thermophilic microorganisms are stable at Proteins from non-thermophilic microorganisms sometimes enter irreversible modifications below 40 ° C. You. BRIEF DESCRIPTION OF THE FIGURES   In the following experimental part, various aspects of the invention will be described with reference to the accompanying drawings. .   FIG. 1 shows the presence of 5M guanidine hydrochloride and 0.25 mM β-mercaptoethanol. The lower hirudin core domain (Hir^1-49) HPLC protocol.   FIG. 2 shows the presence of 3M guanidine hydrochloride and 0.25 mM β-mercaptoethanol. Below is an HPLC protocol for epidermal growth factor.   Experimental procedure   Example 1 Production and Isolation of the Hirudin Core Domain (Hir ^1-49 )   As described by Meyhack et al., Recombinant desulfated hirudin Isolated from Saccharomyces cerevisiae ("T hromb.Res.Suppl. VII, p. 33 (1987)). Desulfato hirudin isolated Was dissolved in 50 mM ammonium bicarbonate pH 8.0 buffer at a concentration of 5 mg / ml, and the solution was dissolved at room temperature for 16 hours. Digest with chymotrypsin (0.25 mg / ml). Trifle digestion to a final concentration of 0.8% Termination is performed by adding oloacetic acid, and the core domain (Hir^1-49) By HPLC Let go.   HPLC conditions:     Column: Vydac C-18     Solvent A: 0.1% trifluoroacetic acid aqueous solution     Solvent B: 0.1% trifluoroacetate acetonitrile solution     Gradient: 10% B to 48% B in 30 minutes at 23 ° C     Hir^1-49Retention time: 14.3 minutes   Example 2: modification of the core domain of Hirudin (Hir ^{1 over 49)}   Fully reduced / denatured hirudin [R], the starting material for the folding experiment The prepared core domain is prepared by the following method.   The core domain of hirudin from Example 1 (2 mg / ml) was replaced with 5 M guanidine chloride. (GdmCl) and 30 mM dithiothreitol, Tris-HCl buffer (0.5 M , PH 8.5). Reduction and denaturation are performed at 23 ° C. for 90 minutes. Open folding To begin, the samples were PD10 equilibrated with 0.1 M Tris-HCl buffer (pH 8.5). (Pharmacia). Desalting takes about 1-2 minutes and the sample is immediately folded Used for folding experiments.   Example 3: Hirudin folding in the presence of guanidine hydrochloride   Dilute the sample to a final protein concentration of 1 mg / ml; 0.1 M Tris-HCl buffer ( pH 8.5) contains 5M guanidine hydrochloride and 0.25mM β-mercaptoethanol doing. After 24 hours of incubation at room temperature, the native hirudin is Separate from the confused hirudin.   HPLC conditions:     Column: Vydac C-18     Solvent A: 0.1% trifluoroacetic acid aqueous solution     Solvent B: 0.1% trifluoroacetate acetonitrile solution     Gradient: 14% B to 32% B in 50 minutes at 23 ° C     Hir^1-49Retention time: 23 minutes   The HPLC-protocol is shown in FIG.   From the integration of the HPLC-protocol values, the original amount of hirudin was 60% ± 5% and K_equilibriumIs Calculated as 0.67 ± 0.15.   The fraction containing the perturbed hirudin is lyophilized, 5M guanidine hydrochloride and 0.1 ml. 0.1 M Tris-HCl buffer containing 25 mM β-mercaptoethanol (pH 8. Dissolve in 5) to a final protein concentration of 1 mg / ml. 24-hour incubation After purification, the original hirudin is separated from the confused hirudin by HPLC as described above. I do. Lyophilization and regeneration are performed a third time as described above.   Combine all fractions containing native hirudin and combine all of the original hirudin The recovery is 97% after 3 cycles.   The activity of the recovered hirudin was converted to human α-thrombin by chromozym (Chromozym, Bo ehringer Manheim) by its ability to not digest. The reaction was 133 mM NaCl and 0.1 67 mM Tris-HCl buffer containing 3% polyethylene glycol 6000 (pH 8.0) at 22 ° C. The rate of digestion was pursued at 405 nm for 2 minutes. Substrate concentration The degree is 200 mM. Adjust the concentration of thrombin to between 2.5 and 25 nM.   For further structural analysis, the recovered protein was washed with Tris-HCl buffer (0.5 M, pH 8.5) with carboxymethylation with 0.2M iodoacetic acid for 30 minutes. Desorb through a PD-10 ™ column equilibrated with a monium solution (50 mM, pH 8.0). Salt. The disulfide content was determined by amino acid analysis (Chang and Knecht "Anal. Biochem.").197 52-58 (1991)) and mass spectrometry (Chatrenet and Chang "J. Biol. Chem. "267, Pp. 3038-3043 (1992)).   Example 4: Hirudin folding in the presence of urea   Except for the only difference in using 8M urea instead of 5M guanidine hydrochloride. If so, regeneration is performed as described in Example 3.   From the integration of the HPLC-protocol values, the original amount of hirudin was 90% ± 5% and K_equilibrium Is calculated as 0.11 ± 0.06.   After two cycles, the total recovery of the original hirudin is 99%.   Example 5: Denaturation of epidermal growth factor   Epidermal growth factor is available from Protein Institute Inc., Broomal l, USA) and modified as described for hirudin in Example 2. .   Example 6: Folding of EGF in the presence of guanidine hydrochloride   Use EGF instead of hirudin and use 5M guanidine instead of 3M guanidine hydrochloride As described in Example 3, except that the only difference was the use of gin hydrochloride. Carry out life.   The HPLC-protocol is shown in FIG.   From the integration of HPLC-protocol values, the original amount of EGF was 89% ± 5% and K_equilibriumIs 0.12 ± Calculated as 0.07.   After two cycles, the total recovery of the original EGF is 99%.   The disulfide content is determined as described in Example 3.

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Claims (1)

[Claims]   1. A method for producing a correctly folded protein or a salt thereof, comprising: The protein is treated with a buffer containing denaturing agents to remove correctly folded proteins. Guanidine hydrochloride at a concentration of 3-7M and 6-10M Wherein said urea is selected from the group consisting of: .   2. Separation of correctly folded proteins, either continuously or discontinuously The method of claim 1.   3. 2. The method of claim 1, wherein the correctly folded protein is discontinuously separated. the method of.   4. 2. The method of claim 1, wherein the protein is hirudin or epidermal growth factor. .   5. 2. The method according to claim 1, wherein the concentration of guanidine hydrochloride is 4-6M.   6. The method according to claim 1, wherein the concentration of urea is 7-9M.   7. The method of claim 1, wherein a reducing agent is present.   8. The method according to claim 7, wherein the reducing agent has a redox potential of 0.20 to 0.30. The described method.   9. The method according to claim 7, wherein the reducing agent is β-mercaptoethanol.   Ten. The method according to claim 7, wherein the concentration of the reducing agent is 0.05 to 1 mM.   11． The method according to claim 7, wherein the concentration of the reducing agent is 0.1 to 0.5 mM.   12． Antibody-based separation, membrane-based separation of correctly folded proteins The method according to claim 1, wherein the separation is performed by electrophoretic separation or chromatographic separation. the method of.   13. Correctly folded proteins can be analyzed by HPLC, TLC or affinity chromatography. The method of claim 1, wherein the separation is by torography.