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
  • C07K14/575—Hormones

Definitions

• the present invention relates to a method for refolding modified human activin A into natural human active human activin A.
• Naturally occurring human activin A is a useful protein that can be expected to be used for pharmaceuticals and the like.
• Back flea technique is a useful protein that can be expected to be used for pharmaceuticals and the like.
• Human activin A is a homodimeric protein consisting of two polypeptide chains of 116 amino acids, isolated and purified from the culture supernatant of human leukemia cell line THP-1 (IF050147). The molecular weight is about 25,000 taltons, and 9 Cys exists per polypeptide chain (18 altogether), a total of 9 intramolecular and intermolecular disulfides Forming a bond. (Biochemical and Biophysical Research Communications, 142, 1095-1103, 1987)
• Human activin A is a culture supernatant obtained after stimulating human myeloid leukemia cells THP-1 (IFO 50147) with Ki-L-Ister (Cell Engineering, Supplement 4, P48-58, 1988). ) Or culture supernatant derived from recombinant CHO cells with high production of human activin A obtained by introducing an expression vector containing human activin A c-DNA.
• denatured human activin A refers to a monomer structure having a polypeptide 'chain of human activin A, but having lost a higher-order structure due to cleavage of intramolecular and Z- or intermolecular disulfide bonds. Or a molecule that has lost its biological activity together with its higher-order structure, such as a molecule in which a disulfide bond has been transferred into a structure different from that of the natural type, or a molecule that has been multimerized by a new intermolecular disulfide bond. .
• modified human activin A Since such modified human activin A has no biological activity, It is necessary to reconstitute the protein into the same higher-order structure as that of the naturally occurring protein. However, in general, some proteins cannot be easily refolded or cannot be refolded at all, and it is very difficult to determine the refolding conditions. In particular, for multimeric proteins and proteins having a large number of disulfide bonds, it is not always possible to set appropriate refolding conditions, and it is not easy to determine refolding conditions.
• Naturally occurring human activin A Since it has a total of 9 intermolecular and intramolecular disulfide bonds, it is extremely difficult to set the conditions for refolding denatured human activin A, and the prior art regarding the method for refolding human activin A There is no.
• J.Mason et al. Examined the relationship between the efficiency of activin A secretion and expression using animal cells and the structure of the expression vector, and found that activin A was refolded and secreted from activin A. It was shown that the prosequence region is essential in addition to the amino acid sequence, and that the amino acid sequence of human activin A alone does not cause refolding or secretion (Science, 247, 1328-1330, 1 990). This report suggests that the refolding of modified human activin A expressed using a microorganism as a production host is extremely difficult.
• An object of the present invention is to provide a method for refolding modified human activin A produced by a microorganism into natural human active human activin A in order to construct an industrial production of human activin A. Disclosure of the invention
• the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that the refolding of denatured human activin A containing the following steps (a) and (b) to natural human activin A having biological activity is carried out.
• Method developed (a) a step of protecting the thiol group of the solubilized modified human activin A, in which all thiol groups are free, with glutathione and / or sodium sulfite;
• the raw material used in the present invention is a modified human activin A culture obtained by culturing a microorganism incorporating the human activin A gene, for example, a recombinant Escherichia coli.
• Escherichia coli When Escherichia coli is used as a production host, most of the human activin A accumulates in the cells as insoluble granules, and these granules may be used as a raw material.
• the insoluble granules of human activin A collected according to a conventional method are suspended in an aqueous solution of low strain (1 to 10 mM) (EDTA (Ethy Ienedi ami netetraacetic acid)), and guanidine hydrochloride as a protein denaturant and Solubilization using Z or urea
• concentration of guanidine hydrochloride and the concentration of urea are generally 4 to 7 M and 6 to 10 M, respectively, which are necessary for denaturing proteins.
• the pH is preferably maintained at 6 or less so as not to form unnecessary bonds. This is completed by stirring at room temperature for 2 to 4 hours.
• a reducing agent is added to the solubilized solution of human activin A at 1 to 100 mM, preferably 5 to 4 OmM, and adjusted to pH 8 to 9 with 20 mM Tris / HCI. After that, it is subjected to a reduction reaction at 20 to 50 ° C for 15 to 360 minutes, and the thiol group, which is the Cys residue, is freed.
• the reducing agent reduced glutachi Compounds such as ON, DTT (Dithiothreitol) 2-mercaptoethanol can be used, but DTT is particularly preferred.
• the addition amount of DTT is preferably 5 to 40 mM.
• Oxidized glutathione is added to the solubilized solution of human activin A, in which all thiol groups are free, to a concentration of 0.05 0.3 M or 0.08 0.16 M, and the mixture is allowed to stand at 0 50 ° C for at least 24 hours.
• Protect groups The thiol group can be protected by sulfonylation using sodium sulfite instead of oxidized glutathione.
• sodium sulfite is added to human activin A in which all of the thiol groups are free so that the final fi concentration becomes 0.05 0.5 M, preferably 0.08 0.16 M, and at the same time, sodium perthionate is added at 520 mM.
• the solution is added to have a concentration of 8 to 16 mM, and the mixture is allowed to stand at 500 ° C. for 2 to 24 hours or more. It is also possible to use a mixture of oxidized glutathione and sodium sulfite.
• the protected state of the thiol group can be confirmed by reverse-phase HPLC using a silica gel column to which a butyl group is chemically bonded, for example, Vydac214TP54 (4.60 x 250 Separations group) (Biochemical and Biophysical Research Communicatons, 142, 1095-1103). , 1987).
• the reagents used in the above operations should be dissolved in a sufficiently degassed solvent before use, and the gas phase of the reaction solution should be filled with inert gas such as nitrogen or helium to suppress air oxidation by oxygen. Is desirable.
• inert gas such as nitrogen or helium
• the refolding buffer used in the present invention is a natural type Any buffer can be used as long as it can maintain the structure of tivin A by 20% or more when denatured by reduction. The details will be described later.
• Methods for replacing protected hactivin A with a refolding buffer include gel-over columns equilibrated with a well degassed refolding buffer, e.g., Sephadex G-25 (Pharmacia Biotech) (provided by Biotech) with thiol group-protected human activin A to recover the protein fraction.
• human activin A which retains the thiol group, is injected into a dialysis membrane, for example, Spectra / Por membrane, No. 1 (manufactured by Spectrum Medical Industries, Inc.), and a sufficiently degassed refolding buffer is injected.
• a dialysis membrane for example, Spectra / Por membrane, No. 1 (manufactured by Spectrum Medical Industries, Inc.)
• a sufficiently degassed refolding buffer is injected.
• a method in which dialysis is carried out at 0 to 20 ° C. for 3 to 24 hours or more with respect to the amount of 500 times or more is also applicable.
• the formation of a disulfide bond of human activin A in which the thiol group is protected is performed by adding a thiol compound.
• the thiol compound is added to the solution containing human activin A, which has been replaced with the refolding buffer as described above, by adding 1 to 10 mM, preferably 2 to 5 mM, and then 0.5 to 50 ° C at 0.5 to 50 ° C. Let stand for 4 days. Completion of refolding and recovery of biological activity can be determined by reversed-phase HPLC and biological activity measurement as described above (eg, measurement of Frent's leukemia cell differentiation induction activity 1 ⁇ , Biochemical and Biophysical Research Communicons, 142, 1095-1103, 1987). Can be confirmed.
• thiol compound thiol compounds such as cysteine, reduced glutathione, DTT, and 2-mercaptoethanol can be used, and cysteine or reduced glutathione is particularly preferable.
• the added amount of cysteine or reduced glutathione is preferably 2 to 5 mM.
• a disulfide bond can be started simultaneously with the replacement with the refolding buffer.
• Hydrochloric acid was added to the thiol-protected human activin A to lower the pH to 2 to 4, Stop the exchange reaction of the bond. Unreacted by dialyzing this against a denaturant, for example, 1 to 100 mM hydrochloric acid containing urea or guanidine hydrochloride, at a volume of 500 times or more at 0 to 20 ° C for 3 to 24 hours.
• the thiol group protecting agent is removed. This is diluted using a refolding buffer that satisfies the conditions described below, and left at 0 to 50 for 0.5 to 14 days.
• the above-mentioned thiol compound is previously added to the refolding buffer in the same manner as described above so as to promote the formation of a disulfide bond.
• One condition of the refolding buffer was determined by the following method. Efficient refolding of human activin A can be achieved by creating a solvent environment that stabilizes the higher-order structure of human activin A, rather than creating a mutant that is convenient for refolding. ⁇ Stability of higher-order structure can be estimated by measuring chemical stability that is considered to correlate with it.
• the chemical stability of human activin A means that there is no change typified by intermolecular association and aggregation and precipitation due to the transfer of disulfide bonds originally formed in the molecule. .
• natural human activin A dissolved in ImM HCl was added to a buffer with a pH of 7.5 to 10.5 consisting of a combination of denaturing agents, surfactants, salts, organic acids, amino acids, sugars, and organic solvents. Dilute. Adjust the strain of natural human activin A to about 0.1 mg Zml. To this is added about 50 equivalents of a quick-acting agent (for example, DTT, 2-mercaptoethanol or arsenic glutathione) with respect to natural type A, and left at room temperature for about 12 hours to perform reverse phase HPLC. Measure the amount of native human activin A remaining.
• a quick-acting agent for example, DTT, 2-mercaptoethanol or arsenic glutathione
• Tris Knuffer urea; 1.5 M, Tris ZHCI; 20 mM, pH 8.5
• Tris buffer urea; 1.5 M, Tris / HC I; 20 mM, pH 8.5
• the ratio of the increase in the residual amount to the residual amount of native human activin A is expressed as the percentage improvement of the residual amount.
• a buffer was prepared by combining various compounds and screened experimentally.
• Tris buffer urea; 1.5 M, Tris ZHCI; 2 0 mM, pH 8.5
• a buffer that can maintain its structure more than 20 o / o more stably was found to be suitable as a refolding buffer.
• the condition is satisfied (the native form of human activin A is reduced and denatured, its structure is lower than that of Tris buffer (urea; 1.5 M, Tris / HCI; 20 mM, pH 8.5)). Can be maintained stably by 20% or more.)
• Any buffer can be used as the refolding buffer used in the present invention. Specifically, the following are examples of refolding buffers that satisfy this condition.
• buffers / cuffers having a buffering action at pH 7.5-10.5 include Tris buffer, trimethylene diamine buffer, and ethanolamine buffer. .
• guanidine hydrochloride 0.05 to 2 M, preferably 0.1 to 1 M can be used in place of the above urea.
• Example 1 Example 1
• the buffer with the highest residual amount was a buffer consisting of (urea: 1.5 M, CHAPS; 2%. Tris / HCI; 2 OmM, pH 8.5). there were.
• Example 1 Using the screened refolding buffer selected in Example 1, the refolding of human activin A was performed.
• oxidized glutathione is added to a final concentration of 0.1 M and added at 5 ° C. Time rested.
• concentration of human activin A was adjusted to 0.1 using a dilution buffer (8 M urea, 2 OmMTris / HCI, pH 8.5). Adjusted to mg / ml. This is a dialysis membrane
• Example 1 In-product and refolding buffer (500 M volume) selected in Example 1 (urea; 1.5 M, 20 mM in Tris / HG, CHAPS; 2%, pH 8.5) ) was dialyzed at 5 ° C for 15 hours.
• Cysteine was finally added to the dialysate to a concentration of 3 mM, and the mixture was allowed to stand at 5 ° C for 3 days. Reconstitution of the higher order structure of human activin A was confirmed by measuring the biological activity (the activity of inducing differentiation of French leukemia cells). as a result, A biological activity (activity of inducing differentiation of French leukemia cells) corresponding to 24% of the amount of the denatured human activin A AA was confirmed.
• Tris / HCI buffer containing 0.13% TDCA / Na (urea; 1.5 M, TDCA / Na; Tris / HCI; 20 ml pH 8.5) were added to each, and these were used in place of the Tris buffer of Example 3 to carry out a reduction reaction at room temperature for 12 hours.
• the residual S of the type hactivin A was examined.
• Table 3 shows the results. The rate of improvement of the residual amount increased with respect to the residual amount when the reduction reaction was performed using Tris / HCI buffer (urine; 1.51 TDCA / Na; 0.13%, Tris / HC and 20 mM; pH, 8.5). The amount is shown by the ratio of the remaining amount in minutes.
• a buffer to which the reagents of Class A or B in Table 1 were added and the reagents of Class AB or C in Table 3 were further added is considered to be more effective as a refolding buffer.
• the reduced human activin A prepared in the same manner as in Example 2 was finally adjusted to 0.1 M with sodium sulfite and finally to 0.01 M with sodium perthionate. And left to stand at 5 ° C for 15 hours.
• the concentration of human activin A was adjusted using a dilution buffer (8 M urea, 20 mM Tris / HCk pH 8.5). Adjusted to 0.1 mg / ml. This was injected into a dialysis membrane (Spectra / Por membrane, No. 1; manufactured by Spectrum Medical Industries, Inc.), and a 500-fold volume of refolding buffer 1 (urea; 1 M, TDC A / Na; 0.5) was used.
• hydrochloric acid was added to lower the pH to 3.0. This is injected into a dialysis membrane (Spectra / Por membrane, Nol; manufactured by Spectrum Medical Industries, Inc.) and contains 500 times the volume of 40 mM hydrochloric acid.
• Buffer 1 urea; 1 M, TDCA / Na; 0.53%, arginine hydrochloride 0.5 M, Tris / HCI; 20 mM, pH 8.5 Buffer 1-2: urea; 1 M, TDC AZNa; 0.53%, sodium chloride; 1 M, Tris / HCI; 20 mM, pH 8.5 Knocker 3: urea; 1 M, TDC AZNa; 0 13%, arginine hydrochloride; 1 M, Tris / HCI; 20 mM, pH 8.5 buffer 14: urea; 1 M, TDCA / Na; 0.13%, arginine hydrochloride; 0.5 M, Tris / HCI; 20 mM, pH 8.5 Buffer
• Buffer 7 Guanidine hydrochloride; 0.5 M, TDC A / Na; 0.5
• Buffer 8 Guanidine hydrochloride; 0.5 M, TDC A / Na; 0.1
• pH 8.5 buffer 9 guanidine hydrochloride; 0.5 M, TDCA / Na; 0.13%, arginine hydrochloride; 0.5 M, Tris / HCI; 20 mM, pH 8.5 Buffer 10: guanidine hydrochloride; 0.5 M, TDC AZNa; 0.13%, calcium sulfate; 0.5 M, Tris / HCI; 20 mM, pH 8.5 Industrial applicability
• the method of the present invention makes it possible for the first time to refold denatured human activin A into native human activin A, and to reduce the biological activity of denatured human activin A by a simple method. You can now recover.
• natural type hactivin A which had previously had to produce eukaryotic cells as a production camp, could be produced simply and inexpensively using recombinant microorganisms as a production camp. .

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• 1996
  • 1996-12-19 EP EP96942573A patent/EP0943690B1/en not_active Expired - Lifetime
  • 1996-12-19 US US09/091,265 patent/US6084076A/en not_active Expired - Lifetime
  • 1996-12-19 JP JP52349797A patent/JP4019432B2/ja not_active Expired - Lifetime
  • 1996-12-19 WO PCT/JP1996/003700 patent/WO1997023638A1/ja not_active Ceased
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