JP2669859B2 - Protein reactivation method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for reactivating a cysteine-containing protein in a reductive denatured state.

BACKGROUND ART Protein is a biopolymer that exerts its specific function by forming a specific higher-order structure (hereinafter referred to as a natural type). The disulfide bond between cysteine residues is also a part of its higher-order structure, and plays an important role in protein function or stability.
Recently, the amount of protein produced has increased due to the development of genetic recombination technology. In particular, in an expression system using Escherichia coli, there are cases where proteins produced in the bacteria are formed as granules [F.A. Marston (FAOMarston)
Et al., Biotechnology (BIO / TECNOLOGY) 2, 800 (198
4), DNBrrms et al., Biochemistry 24 , 7662 (1985)], and the method of collecting proteins from these granules has become an important issue. The protein granules must be denatured once to dissolve. In particular, when a cysteine-containing protein is dissolved, it must be denatured in a reducing state, so in order to reactivate this protein,
A protein in a reductive denatured state must efficiently form a disulfide bond at a position corresponding to the disulfide bond of a native protein. In the conventional technique, the structural change between the denaturing state and the natural type in the presence of this denaturing agent is a two-state transition [protein molecule, page 99-127, Iwanami Shoten (1
985)], it was reactivated by diluting or dialysis in a solution containing no denaturant in one step or in multiple steps [J.A. Gill (JAGill), et al., biotechnology (BIO / tECHNOLOGY) 3, 643 (1985), H. Jay
HJGeorge et al., DN
A) 4, 273 (1985) , JP-A-61-257931]. In this method, the secondary structure and the tertiary structure are formed at the same time, and in the native type, the hydrophobic sites encapsulated in the protein molecule interact with each other, or the natural type is intramolecularly or intermolecularly bound. In many cases, a reaction such as the formation of a disulfide bond occurs at a position different from that of the protein, and the protein does not become a natural type but becomes an aggregate or a denatured form. Particularly, in the case of a protein which has a strong tendency to form an aggregate or a denatured product due to hydrophobic interaction, formation of a disulfide bond at the same position as that of the natural type is suppressed, and only about 1% of the natural type may be obtained.

Therefore, in the conventional reactivation method, it was difficult to efficiently reactivate a cysteine-containing protein from a reductively denatured state, which has a strong tendency to form an aggregate or a denatured body due to a hydrophobic interaction.

Recently, KELangley and co-workers have shown that hydrophobic interactions can occur if they frequently form disulfide bonds at positions corresponding to natural disulfide bonds by forming disulfide bonds in a denatured state. It was reported that the yield of the reactivated protein is improved compared to the case where the formation of aggregates or denatured bodies by the method inhibits the disulfide bond at the position corresponding to the natural type disulfide bond [European Journal. of Biochemistry (Eur.J.Biochem.) 163, 31
3-321 (1987)]. This method relates to the reactivation method of bovine growth hormone. Specifically, after washing bovine growth hormone produced as granules in E. coli, it is solubilized with 6M guanidine hydrochloride and left at room temperature for 20 hours or more to form disulfide bond. Is formed, the gel fractionation is performed in the presence of 6M guanidine hydrochloride, the monomer fraction is separated, diluted to 2M guanidine hydrochloride, and then dialyzed. However, in this method, the yield of the reducing monomer is low when solubilized, and therefore the residual aggregates and the substance derived from the granules that block the SH group of the target protein are at a position corresponding to the natural disulfide bond. Inhibits disulfide bond formation. In addition, since this method is left for 20 hours or more at room temperature during oxidation, it is known that asparagine residues and glutamine residues undergo deamidation under these conditions (Bio-Kimika Biophysica. Actor (Biochi
mica et Biophysica Acta) 214 , 498-508 (1970)].
If the oxidation is performed at a low temperature of about 5 ° C. in order to avoid this, the oxidation process takes a long time.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention provides a method for efficiently reactivating a cysteine-containing protein in a reductive denatured state.

Means for Solving the Problems The present inventors can increase the reducing monomer of a cysteine-containing protein by adding a reducing agent at the same time as adding a denaturing agent to solubilize it, and reduce or eliminate aggregates. Was found.

Furthermore, if the target protein accumulates in the cells of the microorganism as granules, the SH of the target protein can be reduced by adding a reducing agent at the same time as solubilizing the granules by adding a denaturing agent.
When the substance derived from the granules that block the group is released from the target protein and the reducing agent is removed, the substance can be removed together with the reducing agent. As a result, when a disulfide bond is formed in a denatured state, the natural It was found that the yield of a reactivated cysteine-containing protein having a disulfide bond at a position corresponding to the type disulfide bond can be improved, and the present invention has been completed.

The method of the present invention comprises adding a denaturant and a reducing agent to a cysteine-containing protein to solubilize it in a reductive denatured state,
Removal of the reducing agent, oxidation in the denatured state to form a disulfide bond at a position corresponding to the disulfide bond of the native protein, and separation and purification of the reactivated protein by removing the denaturing agent Has become. The present invention is particularly directed to chum salmon growth hormone I.
(SGH-I), eel growth hormone I, etc. It is a poorly soluble cysteine-containing protein that has a strong tendency to form aggregates or denatured substances when it is reactivated by lowering the concentration of denaturants, and has a small number of disulfide bonds. It is suitable for reactivating a protein having no disulfide bond between molecules.

When the protein is accumulated in the cells as granules, the method for separating and purifying the granules is carried out according to the following steps described in JP-A-60-244259.

Next, each step will be described in detail.

<Step 1> Microbial cells accumulating protein granules in the cells are crushed and centrifuged to obtain a precipitate fraction (hereinafter referred to as precipitate fraction A).

For cell disruption, suspend the cells in a buffer solution near neutrality (eg phosphate buffer at pH 7), and then sonicate by a conventional method.
Lysozyme treatment, homogenizer treatment, mechanical pressure can be used. In particular, crushing by mechanical pressure is preferable, and for example, crushing is performed by a French press, Manton-Gaulin homogenizer or the like. Crushing conditions are sufficient in the appropriate use range of each device.

Next, the disrupted liquid is centrifuged to obtain a precipitate fraction A, which contains protein granules and cell residue. Centrifugation may be performed by an ordinary centrifuge, but continuous centrifugation or sharp press may be used. The centrifugation conditions when using a normal centrifuge are usually 2000-15000 rpm, 10-120
Minutes, but 4000 to 12000 rpm and 30 to 90 minutes are particularly preferable.

<Step 2> The precipitated fraction A is converted to an alkali metal or alkaline earth metal mineral salt, pentose, hexose, disaccharide, trisaccharide,
The precipitate is suspended in an aqueous solution of deoxy sugar, sugar alcohol, dextran or dextrin or ficoll, or in an aqueous suspension of Percoll, and centrifuged to obtain a precipitate fraction (precipitated fraction B). By this centrifugation, cell debris is removed as a supernatant, and a precipitate containing protein granules is obtained.

Conventionally, sucrose density gradient ultracentrifugation method, sucrose density gradient equilibrium ultracentrifugation method, etc. are often used to separate intracellular components having different densities. Also known is a fractional centrifugation method in which rat liver is homogenized with a sucrose solution and then centrifuged to obtain a fraction containing cell nuclei in the sedimentation section (Nozomi Ohtake et al., `` Physical Isolation And refining ”, published by the University of Tokyo Press,
Pp. 145-155 (issued in 1976)].

In the above, examples of the alkali metal or alkaline earth metal mineral salts include hydrochlorides such as sodium, potassium, calcium and cesium, sulfates, hydrobromides and the like. Specifically, cesium chloride, calcium sulfate,
Examples include sodium chloride and sodium bromide.

Pentose is L-arabinose, D-xylose, D-ribose and the like, and hexose is D-glycose, D-mannose, D-galactose, L-galactose, D-fructose, L-sorbose and the like, disaccharides,
Trisaccharides include sucrose, maltose, lactose, trehalose, cellobiose, raffinose and the like, deoxy sugars such as L-rhamnose and 2-deoxy-D-ribose, and sugar alcohols such as glycerol, erythritol, arabite, D-sorbitol and D. -Mannitol and the like can be mentioned.

Ficoll and Percoll are trade names of products manufactured by Pharmacia. Ficoll is a water-soluble synthetic polymer consisting of sucrose and epichlorohydrin. Percoll is a synthetic resin made of silica beads. It is coated and the grains are prepared.

The aqueous solution and the aqueous suspension are preferably near neutral buffer solutions.

The concentration of solid components in the aqueous solution is 5 to 50% (w / w in the case of an alkali metal or alkaline earth metal mineral acid salt).
w), 10-80% (w / w) for glycerin, 5-50% (w / w) for dextran and dextrin, 0.25-4M for sucrose and other sugars, especially 0.5- 2M is appropriate.

The ratio of the above-mentioned aqueous solution or water suspension to the precipitation fraction A is 1 / the volume of the fermentation broth before the step 1.
A 20-20 volume (v / v) is appropriate.

The suspension of the precipitation fraction A prepared under the above conditions may be immediately centrifuged to obtain the precipitation fraction B.
In order to enhance the separation efficiency of (1), it is preferable to centrifuge the suspension after sufficiently stirring it.

Centrifugation may be performed by a usual centrifugal separator, but may be performed by continuous centrifugation or a sharp press. In the case of using an ordinary centrifuge, the conditions are usually 2000 to 15000 rpm, 10 to 120 minutes, and preferably 4000 to 12000 rpm, 30 to 90 minutes.

<Step 3> The precipitated fraction B is mixed with a nonionic surfactant or an aqueous solution of cholic acid or its alkali metal salt, and then solid-liquid separated. By this operation, proteins, lipids, lipopolysaccharides, etc. of the membrane components of the microorganisms mixed in the precipitate fraction B are dissolved and removed, and the granules are purified.

In addition, lipopolysaccharide is known as a pyrogen, and it is essential for pharmaceutical production to remove lipopolysaccharide from the granular fraction.

Prior art related to the above method includes Triton X-10.
Schnaitman reported that proteins, lipids and lipopolysaccharides were removed from Escherichia coli membranes by treatment with 0, EDTA (ethylenediaminetetraacetic acid) or the like.

[Journal of Bacteriology (J. Bacteriolo
gy), 108, 553 (1971)].

In the above, the nonionic surfactant is a polyoxyethylene alkyl ether type, for example, polyoxyethylene oleyl ether C ₁₈ H ₃₅ O (CH ₂ CH ₂ O) nH [trade name: Brij 96 (n = 10), Briji 98 (n = 20) etc.], polyoxyethylene fatty acid ester type, for example, polyoxyethylene stearic acid ester [trade name: Nissan Nonion S], polyoxyethylene alkylphenyl ether type, for example, polyoxyethylene pt-octyl Phenyl ether [Brand name: Triton X-100 (n = 9,10), etc.], sorbitan fatty acid ester type, for example, sorbitan monostearate (Brand name: Span 60 etc.), polyoxyethylene sorbitan fatty acid ester type, for example polyoxyethylene monolaurin Sorbitan acid [trade name: Tween 20 (repeat of oxyethylene units = 20), etc.] is used.

Cholic acids or alkali metal salts thereof include cholic acid, deoxycholic acid and their sodium salts.

An aqueous solution of these surfactants (cholic acid and its alkali metal salts are also surfactants) is preferably a buffer solution near neutral.

The concentration of the surfactant in the aqueous solution is 0.2 to 10% (w /
v), preferably 0.5-4%.

The mixed solution of the precipitation fraction B prepared under the above conditions may be subjected to solid-liquid separation immediately, but it is preferable to carry out solid-liquid separation after sufficiently stirring to enhance the separation efficiency.

Solid-liquid separation can be performed by centrifugation, filtration, or the like. Centrifugation may be any method and condition as long as solid-liquid separation is possible. For example, the method and conditions described in steps 1 and 2 may be used.

Purification efficiency can be improved by using a metal chelating agent in combination with the above-mentioned surfactant treatment. As a metal chelating agent, EDTA, EGTA [ethylene glycol bis (2-aminoethyl ether) tetraacetic acid], NTA (nitrilotriacetic acid), HEDTA (2-hydroxyethylethylenediaminetriacetic acid), DTPA (diethylenetriaminepentaacetic acid), DCAT ( 1,2-diaminocyclohexanetetraacetic acid) and the like. The concentration of the metal chelating agent is 2 to 50 mM, preferably 5 to 20 mM. Other conditions may be the same as the case of using the surfactant alone.

In the present invention, as the denaturant, SDS (sodium dodecyl sulfate), urea or guanidine hydrochloride, acid or alkali is used alone or in combination. The type and concentration of the denaturing agent are selected so that the solubilized protein is made as uniform as possible. The reducing agent may be a monovalent thiol compound such as β-mercaptoethanol, cysteine, glutathione, or dithiothreitol (DTT), and is used by adding it to an aqueous solution containing a denaturing agent. DTT with less tendency to form bonds is more preferred.

By adding a reducing agent at the same time that the denaturing agent is added to solubilize the protein, the reducing monomer of the protein can be increased and the aggregates can be reduced or eliminated.
When the target protein accumulates as granules in the microbial cells, a denaturing agent is added to solubilize the granules, and at the same time, a reducing agent is added. It can be released from the protein.

Means for removing the reducing agent include, but are not particularly limited to, dilution under non-oxidizing conditions in the presence of a denaturing agent, dialysis (including ultrafiltration), gel filtration, chromatography including adsorption and desorption, and batch operation, but Gel run is preferred.

In the case of reactivating a protein that accumulates as granules in the microbial cells, the reducing agent can be removed and the substance derived from the granules that block the SH group of the protein can be removed.

Means for oxidation is not particularly limited, in addition to the method by natural oxidation, a method of promoting the oxidation by dissolved oxygen by aerating oxygen or air into the aqueous solution, the oxidation by dissolved oxygen by adding divalent copper ions to the aqueous solution. A method of accelerating by adding, o-iodosobenzoic acid to the aqueous solution,
There is a method of oxidizing by adding a weak oxidizing agent such as oxidized glutathione, a mixture of oxidized glutathione and reduced glutathione, cystine or a mixture of cystine and cysteine. The oxidation rate can be controlled by the pH. In addition, it is desirable that the protein concentration during oxidation is low, and it depends on the protein, but usually 0.1
~ 2000 μg / ml. The reaction time and the reaction temperature are not particularly limited.
The following is desirable. By this oxidation reaction, a protein having a disulfide bond at a position corresponding to a natural disulfide bond is frequently formed.

Means for removing the denaturant include dilution, dialysis (including ultrafiltration), gel filtration, isoelectric focusing, chromatography including adsorption / desorption, and batch operation. Proteins and impurities with disulfide bonds at positions different from the natural type that became the body,
It is removed due to differences in molecular weight, charge, hydrophobicity, etc., and the protein having a disulfide bond at the position corresponding to the natural disulfide bond is separated. Specifically, there are methods such as a) dialysis-centrifugation b) dilution-concentration-isoelectric focusing-centrifugation c) ion exchange chromatography-dialysis-centrifugation d) gel per-ion exchange chromatography.

In each step of the present invention, the reaction temperature is not particularly limited, but is preferably 10 ° C. or lower as long as it does not freeze. The pH of the solution containing the protein varies depending on the protein and is not particularly limited, but pH 2 to 7 is desirable.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention.

Example 1. Method for reactivating SGH-I produced by Escherichia coli from granules (I) Recombinant SGH-I was obtained according to the culture method described in JP-A-61-93197. The cells containing I granules were isolated and purified to a purity of about 90% according to the method disclosed in JP-A-60-244259. 5 mg of this granule was added to 7 M urea and 5 mM EDT.
A, dissolved in 100 mM Tris [Tris (hydroxymethyl) aminomethane] buffer (pH 8.0) containing 1 mM DTT, 2.5 ml
And After stirring at 4 ° C for 2 hours, centrifuge (12,000 rpm,
After 5 minutes), DTT was removed from the supernatant using a gel per column PD-10 (Pharmacia Fine Chemical Co.) equilibrated with 100 mM Tris buffer (pH 7.0) containing 7 M urea. 3.5 ml of the protein eluate fraction was mixed with an equal amount of 7 M urea and 100 mM Tris buffer (pH 9.0) containing 1 mM CuSO ₄ , and the mixture was allowed to stand at 4 ° C. for 2 hours to promote oxidation. Next, 7 ml of this solution was added to 2 100 mM Tris buffer (pH
7.0) at 4 ° C overnight. Centrifuge the dialysate (12,
000 rpm, 5 minutes), the supernatant was almost pure recombinant SG
HI was recovered. According to the method of Sekine et al. [Proceeding of the National Academy of Science (Proc.Natl.Acad.Sci.) 82 4306 (1985)],
When the activity of the obtained recombinant SGH-I was measured, natural SGH-I [H. Kawauchi et al., Archives of Biochemistry and Biophysic
s) 244 542 (1986)]. Table 1 shows the material balance of the process.

Example 2 Purification Method of Natural SGH-I after Oxidation Step (I) The solution of the oxidized solution obtained in Example 1 was diluted 10-fold with 100 mM Tris buffer (pH 8.0) and heated to 4 ° C. And left for 2 hours, using an ultra-membrane (YM-10, manufactured by Amicon),
Concentrated to zero. After adjusting the pH of the concentrated solution to 6.0 with 1N hydrochloric acid to cause isoelectric point precipitation, centrifugation (12,000 rpm, 5 minutes) was performed to recover almost pure recombinant SGH-I in the supernatant. . Sekine et al.'S method [Proceeding of the
National Academy of Science (Proc.Nat
l.Acad.Sci.) 82 4306 (1985)], the activity of the obtained recombinant SGH-I was measured.
[H. Kawauchi et al., Archives of Biochemistry and Biophysics 244 54
2 (1986)]. Table 2 shows the material balance of the process.

Example 3. Purification method of natural SGH-I after oxidation step (I
I) The solution after oxidation obtained in Example 1 was diluted 10-fold with 7 M urea, adjusted to pH 8.0, and then equilibrated with 10 mM Tris buffer (pH 8.0) containing 7 M urea. DEAE Sepharose CL-6B (Pharmacia Fine Chemicals)
The solution was passed through a column (1.5 × 5.7 cm) (10 ml / hour). After washing with 50 ml of the same buffer (10 ml / hour), wash with 30 ml of the same buffer.
Elution (5 ml / hour) was performed by a concentration gradient method using 10 mM Tris buffer (pH 8.0) containing M urea and 1 M NaCl. The eluate is 1
When fractionation was performed with 5 ml, almost pure recombinant SGH-I could be recovered in the third fraction (5 ml). Next, 5 ml of this solution was dialyzed against 1.5 100 mM Tris buffer (pH 8.0) at 4 ° C. overnight. Centrifuge the dialysate (12,000
After 5 minutes (rpm), recombinant SGH-I having the same three-dimensional structure as natural SGH-I including the position of disulfide bond was recovered in the supernatant. Sekine et al.'S method [Proceding of the National Academy of Sciences (Proc. Natl. Acad. Sci.) 82 4306 (1985)]
When the activity of the obtained recombinant SGH-I was measured by the method, the natural SGH-I [H. Kawauch (H. Kawauch
i) et al., Archives of Biochemistry a
nd Biophysics) 244 542 (1986)]. Table 3 shows the material balance of the process.

Example 4. Reactivation method from eel growth hormone I granules produced by E. coli Recombinant eel growth hormone I granules were obtained by the following method. First, Escherichia coli EUPA1 (FERM B containing the recombinant plasmid pUPA1
P-825) in 10 ml of MCG medium [0.6% Na ₂ HPO ₄ , 0.3% KH ₂ PO
₄ , 0.5% NaCl, 0.1% NH ₄ Cl, 0.5% glucose, 0.5% casamino acid, 1 mM MgSO ₄ , 4 μg / ml vitamin B ₁ , pH 7.2] and inoculated at 30 ° C. for 7 hours. 50 ml of the obtained culture solution
G medium was inoculated and cultured at 30 ° C. for 18 hours. The resulting culture solution was inoculated into 1 MCG medium and cultured at 30 ° C. for 5 hours.
Culturing was carried out at ℃ for 2 hours and further at 37 ℃ for 41 hours. The obtained culture solution was centrifuged at 8,000 rpm for 10 minutes to collect bacterial cells. The resulting cells were isolated and purified according to the method disclosed in JP-A-60-244259 to obtain eel growth hormone I granules having a purity of about 90%. 5 mg of this granule is treated with 7 M urea, 5
100 mM Tris buffer (pH 8.0) containing mM EDTA and 1 mM DTT
To 2.5 ml. After stirring at 4 ° C for 2 hours, centrifuge (12,000 rpm, 5 minutes) and add 7M urea from the supernatant.
The reducing agent was removed using a gel per column PD-10 (Pharmacia Fine Chemical Co.) equilibrated with 00 mM Tris buffer (pH 7.0). The protein elution fraction (3.5 ml) was mixed with an equal amount of 7 M urea and 100 mM Tris buffer (pH 9.0) containing 1 mM CuSO ₄ , and the mixture was allowed to stand at 4 ° C. for 2 hours to promote oxidation. Next, 7 ml of this solution was dialyzed against 2 100 mM Tris buffer (pH 7.0) at 4 ° C. overnight. When the dialyzed solution was centrifuged (12,000 rpm, 5 minutes), almost pure recombinant eel growth hormone I was recovered in the supernatant. Radioimmunoassay [M. Kishida et al., General and Comparative Endocrinology 65 478
(1987)] and the binding activity to the antibody was measured, the natural eel growth hormone I [M. Kishida]
Et al., General and Comparative Endocrinology 6
5 478 (1987)] and has the same biological activity as the natural type. Table 4 shows the material balance of the process.

Example 5. Reactivation method from granules of SGH-I produced by Escherichia coli (II) Recombinant SGH-I was a recombinant SGH-I obtained according to the culture method disclosed in JP-A-61-93197. The cells containing I granules were isolated and purified to a purity of about 90% according to the method disclosed in JP-A-60-244259. 5 mg of this granule was added to 7 M urea and 5 mM EDT.
A, 2.5 mM was dissolved in 100 mM Tris buffer (pH 8.0) containing 1 mM DTT. After stirring at 4 ° C for 2 hours, centrifugation (12,
Gel per column PD-1 equilibrated with 100 mM Tris buffer (pH 7.0) containing 7 M urea from the supernatant after 5,000 rpm for 5 minutes)
0 (Pharmacia Fine Chemical Co., Ltd.)
Was removed. Oxidation was allowed to proceed by mixing 3.5 ml of the protein eluted fraction with an equal amount of 7 mM urea and 100 mM Tris buffer (pH 8.0) containing 0.2 mM oxidized glutathione, and allowing it to stand at 4 ° C. overnight. . Then add 7 ml of this solution to 1 of 2
It was dialyzed against 00 mM Tris buffer (pH 7.0) at 4 ° C. overnight. When the dialyzed solution was centrifuged (12,000 rpm, 5 minutes), almost pure recombinant SGH-I was recovered in the supernatant. Sekine et al. [Proceeding of the National Academy of Science (Proc.Natl.Acad.Sci.) 82]
4306 (1985)], the activity of the obtained recombinant SGH-I was measured, and natural SGH-I [H. Kawauchi et al., Achievements of Biochemistry and Biophysics ( Archives of Bi
ochemistry and Biophysisc) 244 , 542 (1986)]. Table 5 shows the material balance of the process.

Reference Example 1 Salmon growth hormone I using a conventional method
Reactivation method of SGH-I granules obtained in the same manner as in Example 1
It was dissolved in 100 mM Tris buffer (pH 8.0) containing urea, 5 mM, EDTA and 1 mM DTT to make 12.5 ml. After stirring at 4 ° C for 2 hours, the mixture was centrifuged (12,000 rpm, 5 minutes), and the supernatant was diluted to 3: 1.
It was dialyzed against 00 mM phosphate buffer (pH 7.0) at 4 ° C. overnight. The dialyzed solution was centrifuged (12,000 rpm, 5 minutes) to obtain a supernatant. Table 6 shows the material balance of the process. Almost pure recombinant SGH-I was recovered in the supernatant obtained by centrifuging the dialysis solution, but the yield was very low and not practically effective.

Reference Example 2 Reactivation Method of Eel Growth Hormone I Using Conventional Method 5 mg of recombinant eel growth hormone I granules obtained in the same manner as in Example 4 was added to 100 mM containing 7 M urea, 5 mM EDTA and 1 mM DTT. It was dissolved in Tris buffer (pH 8.0) to make 12.5 ml. After stirring at 4 ° C for 2 hours, centrifugation (12,000 rpm, 5 minutes) was performed, and the supernatant was added to 3 of 100 mM phosphate buffer (pH 7.0).
Dialyze at ℃ overnight, centrifuge the dialysate (12,000 rpm, 5
Then, a supernatant was obtained. Table 7 shows the material balance of the process. Almost pure recombinant eel growth hormone I was recovered in the supernatant obtained by centrifuging the dialyzed solution, but the yield was very low and not practically effective.

Reference Example 3 Method for reactivating chum salmon growth hormone I by the method of KELangley et al. [Eur. J. Biochem 163 , 313-321 (1987)].

100 mg of SGH-I granules obtained in the same manner as in Example 1
It was dissolved in 6 M guanidine hydrochloride and 50 mM Tris buffer (pH 8.0) to make 10 ml. After standing for 80 hours, the solution was passed through a Sephacryl S-200 (Pharmacia Fine Chemical Co.) column (2.6 × 94 cm) equilibrated with the above buffer at 50 ml / hour. Subsequently, it was eluted with the same buffer. The eluate was fractionated with 5 ml as one fraction to obtain an elution fraction of 35 ml. This eluted fraction
0.25% (w / v) NaHCO ₃ , 0.2% (w / v) α-lactose,
0.2% (w / v) mannitol (pH 8.5) was added to make 105 ml. It was subsequently dialyzed against 30 of the above buffers at room temperature for 24 hours. The supernatant obtained by centrifuging the dialysate was obtained. Table 8 shows the material balance of the process. Almost pure recombinant SGH-I was recovered in the supernatant obtained by centrifuging the dialyzed solution, but the yield was low and not practically effective.

Effects of the Invention According to the present invention, a cysteine-containing protein in a reductive denatured state can be efficiently reactivated.

Claims (7)

(57) [Claims] 1. A solubilizing agent in a reduced denaturing state by adding a denaturing agent and a reducing agent to a sparingly soluble cysteine-containing protein,
The reducing agent is removed, and the denaturing agent is oxidized in a denatured state without diluting to form a disulfide bond at a position corresponding to the disulfide bond of the native protein, and the denaturing agent is reactivated by removing the denaturing agent. A method for reactivating a protein, which comprises separating and purifying the activated protein. 2. The method according to claim 1, wherein the sparingly soluble cysteine-containing protein is a protein produced as granules in the cells of a microorganism. 3. The method according to claim 1, wherein the denaturing agent is SDS, urea, guanidine hydrochloride, acid or alkali. 4. The method for removing a reducing agent in the presence of a denaturing agent by chromatography under non-oxidizing conditions, dialysis, ultrafiltration, gel filtration, adsorption / desorption, or batch operation. Alternatively, the method described in (2). 5. The method according to claim 1 or 2, wherein the oxidizing means is selected from an air oxidizing method by aeration, an air oxidizing method using a metal ion as a catalyst, and an oxidizing method using a weak oxidizing agent. 6. The method according to claim 1, wherein the weak oxidizing agent is o-iodosobenzoic acid, oxidized glutathione, a mixture of oxidized glutathione and reduced glutathione, cystine or a mixture of cystine and cysteine. . 7. The protein concentration during oxidation is 0.1 to 2000 μg / m.
The method according to claim 1 or 2, wherein l is l.