JP4625433B2 - Protein refolding agent and refolding method - Google Patents

Description

  The present invention relates to a protein refolding agent and a refolding method, and more particularly to a refolding agent and a refolding method used to unwind an unfolded protein into an active protein structure.

  Elucidation and analysis of protein functions and structures are extremely important, for example, directly related to disease treatment and drug discovery. For this reason, various proteins are synthesized and produced by various methods, their structures are examined, and action mechanisms and roles in the living body are elucidated. Now, it is well known that the functions of proteins are determined not only by the sequence and chain length of the amino acids constituting them, but also by the ordered three-dimensional structure (higher order structure) taken by them.

Industrially, with the development of genetic engineering, various proteins have been prepared in large quantities as recombinants and used in a wide variety of industries such as pharmaceutical manufacturing, food processing, and clinical diagnosis. With the development of vector technology, technology that produces a large amount of target protein in Escherichia coli and yeast cells can be easily and reproducibly executed with few resources.
However, many of the proteins expressed in recombinants are unordered in their three-dimensional structure, their higher-order structures are not controlled, and often form small particle granules called inclusive inclusion bodies (inclusion bodies). . For this reason, in the production process by E. coli, the inclusion body is unwound (unfolded), the higher-order structure is prepared, and the protein is converted into a soluble protein with an ordered three-dimensional structure, that is, the inclusion body is unfolded and then refolded. It is necessary to (rewind).

  This type of refolding is an extremely important technique that can be applied not only to proteins produced by E. coli and yeast, but also to the regeneration of proteins inactivated due to certain causes such as thermal history. Therefore, this refolding has been extensively studied and various methods have been proposed, but most of them have a low refolding rate and a certain limited protein (especially a specific protein having a low molecular weight). In many cases, this refolding is general, universal, and has a high refolding rate applicable to various proteins. It is not an efficient and economical method.

The refolding operation that has been used for a long time is the dialysis method and the dilution method. In the dialysis method, a protein denaturing agent (an unfolding agent such as guanidine hydrochloride and / or urea) added in advance by dialysis is gradually diluted and replaced with a buffer solution to obtain a functional protein three-dimensional structure. It is a method of refolding. As an application example of this method, a stepwise dilution method is known in which the yield of refolding to a functional protein tertiary structure is increased by further slowly decreasing the protein denaturant concentration (for example, non-patented). Reference 1).
However, the dialysis method is not practical in industry because the external solution usually requires a volume of 100 times or more of the protein solution and requires several days of time.

The dilution method is widely used because it ends in a relatively short time and requires a relatively small volume compared to the dialysis method. In the dilution method, an unfolded protein solution to which a protein denaturant (unfolding agent) is added is excessively diluted with a buffer solution to refold the protein from an unfolded state to a functional protein structure. It is a method to do. The dilution method is the simplest and low-cost method for refolding a protein into a functional three-dimensional structure, but the yield of refolding is poor (for example, Non-Patent Document 2), and the yield is low due to large dilution. is the current situation.

  The use of an adsorption separation column for refolding has also been tried. When protein or thioredoxin unfolded with urea / guanidine hydrochloride is subjected to gel filtration, the refolding occurs during gel filtration (Non-patent Document 3). However, refolding is not always sufficient with this method, and other proteins usually do not give satisfactory results. When a protein that has been solubilized with 8M urea is adsorbed on a column that is fixed with a molecular chaperone GroEL, which is a kind of protein that promotes refolding of a protein with a broken structure, and eluted with a solution containing 2M each of potassium chloride and urea, It has also been reported that refolding of the eluted protein occurs (Non-patent Document 4). However, these are only recognized with very limited proteins such as Cyclophilin A. In particular, when a molecular chaperone is used, since it is a certain type of template, the fact that it does not conform to the shape of the template is completely useless.

  In some cases, a metal chelate is used instead of a protein that promotes refolding as a fixed substance on the column. When a His6-tag fusion protein unfolded in an aqueous solution containing guanidine hydrochloride and urea is adsorbed on a resin to which a nickel chelate has been immobilized and washed with a buffer solution containing no unfolding agent, refolding of the fusion protein occurs (non-folding) Patent Document 5). The application of this method is limited to this protein, and the resin preparation is complicated and expensive.

When β-cyclodextrin or cycloamylose is used as an artificial chaperone and a protein unfolded with a surfactant is mixed into this chaperone solution, the surfactant is taken up by the artificial chaperone, and the protein is refolded in this process. There are also reports (Non-Patent Documents 6 to 8). However, this method is only successful with carbonic anhydrase B and the like. Moreover, it is not a method that can be repeated, but is expensive.
J Biol Chem. 2003 Mar 14; 278 (11): 8979-8987. J Immunol Methods. 1998 Oct 1; 219 (1-2): 119-129. Biochemistry, Vol. 26 (1987) 3135-3141 Natl. Acad. Sci. USA, Vol. 94 (1997) 3576-3578 Life Science News (Japan Ed.) Vol. 3 (2001) 6-7 J. et al. Am. Chem. Soc. Vol. 117 (1995) 2373-2374 J. et al. Biol. Chem. Vol. 271 (1996) 3478-3487 FEBS Lett. Vol. 486 (2000) 131-135

As described above, various refolding methods have been proposed, but when refolding an unfolded protein, there were still problems such as a decrease in yield and low purity due to large dilution of the protein. .

As a result of intensive studies to solve the above problems, the present inventors have reached the present invention.
That is, the present invention relates to a method for refolding an unfolded protein, comprising a group consisting of phosphate, pyrophosphate, polyphosphate, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and salts thereof. Ri Do the step of treatment with one or more compounds selected (a) Tona Ru refolding agent (C), the concentration in the system of the refolding step (a) is at 0.5-6 mol / L A refolding method; and a method for producing a protein comprising the steps of refolding by the above method.

  When the refolding agent of the present invention is used, productivity does not need to be greatly diluted, so that productivity is dramatically improved as compared with the conventional case. Moreover, since the refolding effect is higher than before, a high-purity protein can be obtained in large quantities.

  The refolding agent of the present invention is an unfolded protein refolding agent, and comprises a phosphorus-containing refolding agent (A) having a group represented by the following general formula (1) (A) C).

  P in the general formula (1) is a phosphorus atom, and examples of the compound (A) include inorganic phosphoric acid and a salt thereof (A1); an alkyl phosphoric acid ester and a salt thereof (A2); (A3) and the like.

Inorganic phosphoric acid and its salt (A1) include phosphoric acid, hypophosphorous acid, phosphorous acid, hypophosphoric acid, pyrophosphoric acid, pyrophosphorous acid, metaphosphoric acid, tripolyphosphoric acid, polyphosphoric acid (tetrapolyphosphoric acid, hexa Polyphosphoric acid and the like), and salts thereof.
Salts include alkali metal salts (sodium salt, potassium salt, lithium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, barium salt, etc.), ammonium salt, amine salt (primary amine salt, secondary amine salt). Tertiary amine salts), and quaternary ammonium salts (such as tetraalkylammonium salts).
Specific examples of the salt of (A1) include phosphoric acid monohydrogen disodium salt, phosphoric acid dihydrogen monosodium salt, phosphoric acid monohydrogen dipotassium salt, phosphoric acid dihydrogen monopotassium salt, ammonium phosphate salt, Phosphate salts such as tetramethylammonium phosphate, tetraethylammonium phosphate, triethylamine phosphate and triethanolamine phosphate; sodium phosphite, potassium phosphite, ammonium phosphite salt, tetraphosphite tetra Phosphites such as methylammonium salt, tetraethylammonium phosphite, triethylamine phosphite and triethanolamine phosphite; sodium pyrophosphate, potassium pyrophosphate, tetramethylammonium pyrophosphate, pyrophosphoric acid Tetraethylammonium salt, triethyla pyrophosphate Emissions salts and pyrophosphoric acid triethanolamine salt; sodium tripolyphosphate, potassium tripolyphosphate, and tripolyphosphate salts such as tripolyphosphate tetraethylammonium salts.

Examples of the alkyl phosphate esters and their salts (A2) include alkyl phosphate esters (A21), alkyl pyrophosphate esters (A22), and alkyltripolyphosphate esters (A23) having an alkyl group having 1 to 12 carbon atoms. It is done.
Examples of the alkyl phosphate ester (A21) having an alkyl group having 1 to 12 carbon atoms include methyl phosphate ester, ethyl phosphate ester, propyl phosphate ester, 2-propyl phosphate ester, butyl phosphate ester, and 2-butyl phosphate. Examples include esters, t-butyl phosphate, pentyl phosphate, hexyl phosphate, cyclohexyl phosphate, octyl phosphate, nonyl phosphate, decyl phosphate, dodecyl phosphate, and the like.
As the alkyl pyrophosphate ester (A22) having an alkyl group having 1 to 12 carbon atoms,
For example, methyl pyrophosphate ester, ethyl pyrophosphate ester, propyl pyrophosphate ester, 2-propyl pyrophosphate ester, butyl pyrophosphate ester, 2-butyl pyrophosphate ester, t-butyl pyrophosphate ester, pentyl pyrophosphate ester, hexyl pyroline Examples include acid esters, cyclohexyl pyrophosphate esters, octyl pyrophosphate esters, nonyl pyrophosphate esters, and decyl pyrophosphate esters.
Examples of the alkyltripolyphosphate (A23) having an alkyl group having 1 to 12 carbon atoms include methyltripolyphosphate, ethyltripolyphosphate, propyltripolyphosphate, 2-propyltripolyphosphate, butyltripolyphosphate, 2 -Butyltripolyphosphate, t-butyltripolyphosphate, pentyltripolyphosphate, hexyltripolyphosphate, cyclohexyltripolyphosphate, octyltripolyphosphate, nonyltripolyphosphate, decyltripolyphosphate, and the like.
Examples of the salts of (A21) to (A23) include the same alkali metal salts, alkaline earth metal salts, ammonium salts, amine salts and quaternary ammonium salts as the salts mentioned in the above (A1).

  Sugar phosphates and their salts (A3) include adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and cyclic adenosine monophosphate (c-AMP) and its sodium Salt, potassium salt, triethylamine salt, triethanolamine salt and the like can be mentioned.

  Among the phosphorus-containing compounds (A), (A1) and (A3) are preferable from the viewpoint of the refolding effect, and more preferable are phosphoric acid, pyrophosphoric acid, polyphosphoric acid, adenosine triphosphate, and adenosine 2-phosphorus. Acids, adenosine monophosphate and their salts.

The refolding agent (C) of the present invention is used for refolding an unfolded protein. In the step of treating this protein with a refolding agent, the refolding agent (C) of the present invention is usually used at a concentration of 0.2 to 6 mol / L as the concentration of the phosphorus-containing compound (A) in the system.
The concentration of (A) in the system is usually 0.2 mol / L or more, preferably 0.3 mol / L or more, more preferably 0.5 mol / L or more. By being in this concentration range, the effect | action which returns the structure of the unfolded protein to a normal structure can be expressed effectively.

  In addition, in the refolding agent of this invention, water is contained normally, and the below-mentioned surfactant (D), pH adjuster (E), and protein stabilizer (F) can be further contained as needed. .

The unfolded protein in the present invention may be a protein unfolded by any method, but from the viewpoint of the refolding effect, a protein unfolded by guanidine hydrochloride, urea or a combination thereof is preferable. Guanidine, urea, or a protein unfolded in an aqueous solution having a total concentration of 0.5 mol / L or more is usually used.
When the protein contains a S—S bond in the molecule, in addition to guanidine hydrochloride and / or urea unfolding agent, reduction of 2-mercaptoethanol, dithiothreitol, cystine or thiophenol The protein may be unfolded by adding an agent.

The molecular weight of the unfolded protein in the present invention is 1,000 to 300,000, and preferably 10,000 to 250,000 from the viewpoint of the refolding effect.
In general, there is a correlation between the molecular weight and the difficulty of refolding, and it is said that refolding becomes extremely difficult when the protein has a large molecular weight (molecular weight of about 10,000 or more). Since the refolding method of the present invention has an excellent refolding effect, it is a very effective method even for high molecular weight proteins having a molecular weight of 10,000 or more.
Since proteins having a molecular weight of less than 1,000 can be easily rewound, the refolding method of the present invention works particularly effectively on proteins having a molecular weight of 1,000 or more. On the other hand, if the molecular weight exceeds 300,000, the solubility in water is remarkably lowered, which is not preferable.
The molecular weight of the protein can be measured by general gel electrophoresis.

The refolding method of the present invention is a method for refolding an unfolded protein, and includes a step of treating the protein with the above-described refolding agent, in which the phosphorus-containing refolding agent (C) is contained in the system. The concentration of compound (A) is usually 0.2 to 6 mol / L.
The phosphorus-containing refolding agent (C) has a concentration of (A) in the system of usually 0.2 mol / L or more, preferably 0.3 mol / L or more, more preferably 0.5 mol / L or more. is there. By being in this concentration range, the effect | action which returns the structure of the unfolded protein to a normal structure can be expressed effectively.
When the concentration of (A) is too low, the refolding effect (ratio at which the unfolded protein can be refolded) is poor.
In addition, “productivity”, which is an evaluation item in Examples described later, is a product of “enzyme activity” and a dilution factor, and represents a so-called yield.

The phosphorus-containing compound (A) in the present invention is such that when the concentration in the system is 0.2 mol / L or more, a hydrogen bond between the group represented by the general formula (1) and the protein is easily formed. Estimated to reach equilibrium concentration.
Conventionally, in the refolding process, a general phosphoric acid compound (for example, phosphoric acid / sodium phosphate, etc.) may be used as a buffering agent. / L or less, and at most 0.05 mol / L or less, no refolding action was observed.
In addition, if the concentration of the phosphorus-containing compound (A) in the system is too high, the viscosity of the system tends to increase, which makes it difficult to separate and purify the protein in the subsequent protein production step, which is not preferable.

  The step of treating the protein with the refolding agent in the present invention is a step of stirring and mixing the protein and the refolding agent to such an extent that the heterogeneous portion is eliminated, and then, the refolding is further advanced sufficiently. In addition, if necessary, it may be allowed to stand for a certain period of time. The standing time is, for example, 1 to 50 hours.

The refolding method of the present invention may further use a surfactant (D) in the step of treating with a refolding agent.
Examples of the surfactant (D) include the following nonionic surfactants, cationic surfactants, anionic surfactants and amphoteric surfactants. The addition of a surfactant is preferred from the viewpoint of suppressing protein aggregation.

Nonionic surfactants include higher alcohol alkylene oxide (hereinafter abbreviated as AO) adducts (higher alcohols having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc.)). Ethylene oxide (hereinafter abbreviated as EO) of 1 to 20 mol, etc.], alkylphenol AO adduct having 6 to 24 carbon alkyl, polypropylene glycol EO adduct and polyethylene glycol PO adduct, pluronic type surface activity Agents, fatty acid AO adducts, polyhydric alcohol type nonionic surfactants and the like.
Examples of the cationic surfactant include a quaternary ammonium salt type cationic surfactant and an amine salt type cationic cationic surfactant.
Examples of the anionic surfactant include ether carboxylic acid or a salt thereof, sulfate ester or ether sulfate ester and salts thereof, sulfonate, sulfosuccinate, fatty acid salt, acyl having a hydrocarbon group having 8 to 24 carbon atoms. And naturally occurring carboxylic acids and salts thereof (eg, chenodeoxycholic acid, cholic acid, deoxycholic acid, etc.).
Examples of amphoteric surfactants include betaine-type amphoteric surfactants and amino acid-type amphoteric surfactants.
Examples of the surfactant (D) include surfactants described in JP-B-57-39678 in addition to the above.

The surfactant (D) in the refolding method of the present invention is preferably a nonionic active agent from the viewpoint of little interaction with the protein.
Although it does not specifically limit as a nonionic surfactant, TWEEN20, TWEEN40, TWEEN60, TWEEN80, Triton-X100, Triton-X300 is mentioned as what can be obtained as a commercial item.

  The addition amount of the surfactant (D) is usually 20% or less with respect to the addition amount of 100% by weight of (A), preferably 0.001 to 20% from the viewpoint of the refolding effect, and 0.01 to 5% is more preferable.

The refolding method of the present invention may further use a pH adjuster (E) and a protein stabilizer (F) in the step of treating with the refolding agent.
As the pH adjuster (E), Tris (N-tris (hydroxymethyl) methylaminoethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), and phosphate buffer (For example, monosodium dihydrogen phosphate + aqueous hydrochloric acid solution or monosodium dihydrogen phosphate + sodium hydroxide aqueous solution).
In addition, when using a phosphate buffer, since a phosphate buffer is a phosphate, it may overlap with (A) in this invention partially. However, as described above, the upper limit of the addition concentration of the phosphate buffer used for the purpose of pH adjustment is 0.05 mol / L at the highest, and the phosphorus-containing compound (A) as the refolding agent of the present invention When the pH adjustment is used together with the target phosphate buffer, the concentration in the system of (A) is preferably 0.2 to 5.95 mol / L.

  In general, the refolding operation is performed at pH 7 to 8. When the pH adjuster (E) is added, the amount added is not particularly limited as long as it is adjusted within this range, but the addition of (A) The amount is usually 20% or less with respect to the amount of 100% by weight, preferably 0.001 to 20% by weight, and more preferably 0.01 to 20% by weight from the viewpoint of the refolding effect.

Examples of the protein stabilizer (F) include reducing agents, polyols, metal ions, chelating reagents and the like.
Examples of the reducing agent include 2-mercaptoethanol, dithiothreitol, ascorbic acid, reduced glutathione and cysteine.
Examples of polyols include glycerin, glucose, sucrose, ethylene glycol, sorbitol and mannitol.
Examples of metal ions include divalent metal ions such as magnesium ions, manganese ions, and calcium ions.
Examples of the chelating reagent include ethylenediaminetetraacetic acid (EDTA) and glycol etherdiamine-N, N, N ′, N′-4 acetic acid (EGTA).

  When the protein stabilizer (F) is added, the amount added is usually 10% by weight or less with respect to 100% by weight of (A), and 0.001 to 10% from the viewpoint of the refolding effect. % Is preferable, and 0.01 to 5% by weight is more preferable.

In the refolding method of the present invention, if the protein concentration in the system exceeds a certain amount, the viscosity in the system becomes too high, and the enzyme activity decreases, which is not preferable. On the other hand, since the production efficiency depends on the product of the concentration of the protein in the system and this enzyme activity, the production efficiency depends on the concentration of the protein in the system in the low concentration region of the protein system. Decreases in the area. As a result, from the viewpoint of protein production efficiency, the protein concentration in the system is preferably 0.2 to 30 mg / mL, more preferably 0.2 to 20 mg / mL, and particularly preferably 0.25 to 5 mg / mL. It is.
The term “in the system” as used herein refers to the refolding agent, unfolding agent, water, other optional surfactant (D), pH adjuster (E), and protein stabilizer (F) as a whole. means.
Moreover, the addition amount (weight) of (A) with respect to the weight of the protein is preferably 5 to 2,000 parts with respect to 1 part of the protein, and more preferably 10 to 1,000 parts from the viewpoint of the refolding effect. .

The protein production method of the present invention is a protein production method including the step of refolding by the above refolding method.
Since the protein obtained by the protein production method of the present invention is obtained by the above refolding method, it has a higher purity than the conventional method, and it is not necessary to carry out a large dilution, so that a high yield can be obtained.
Examples of the protein production method of the present invention include a production method according to the following sequence of steps.
(1) Protein culturing step: Enzyme or recombinant protein is cultured in a protein producer such as E. coli.
(2) Lysis process: The inclusion body in the protein producing body is removed by using a lysing agent or the like.
(3) Unfolding step: 0.5 mol / L or more of an unfolding agent and, if necessary, a reducing agent of 20 mmol / L or less are added to an inclusion body suspension (for example, 10 mg protein / mL), and the mixture is gently stirred. Leave for a few hours.
(4) Refolding step: Add (C) to the unfolded protein suspension, stir lightly, and leave at room temperature overnight to perform refolding.
(5) Separation / removal step: The target normal protein is separated from the suspension by column chromatography or the like.

Examples of the protein producer in the protein culturing step (1) include the following bacterial cells.
Bacterial cells include streptococci, staphylococci, Escherichia, streptomyces and Bacillus cells, fungal cells such as yeast cells and Aspergillus Genus (Aspergillus) cells, insect cells: eg Drosophila S2 (Spodoptera S2), Spodoptera Sf9 (
Spodoptera Sf9) cells, animal cells: for example, CHO, COS, Hela, C127, 3T3, BHK, 293, and Bowes melanoma cells, plant cells, and the like.
As a specific example of the genus Escherichia (Escherichia), Escherichia coli (E. coli) K12DH1 [Proc. Natl. Acad. Sci. 60, 160 (1968)], JM103 (see Nucleic Acids Research 9, 309 (1981)), JA221 [Journal of Molecular Biology (Journal) of Molecular Biology) 120, 517 (1978)], HB101 [Journal of Molecular Biology 41, 458] See 1969)], C600 [Genetics (Genetics) 39 vol, see 440 pp (1954)], MM294 [Nature (Nature) 217 Vol see 1110 pages to (1968)] and the like.
Specific examples of Bacillus include Bacillus subtilis MI114 [see Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry 95]. Volume, p. 87 (1984)].

As a recombinant protein culturing method, an expression vector containing cDNA encoding the target protein is:
(I) A messenger RNA (mRNA) is isolated from the target protein-producing cell, a single-stranded cDNA and then a double-stranded DNA are synthesized from the mRNA, and the complementary DNA is incorporated into a phage or a plasmid.
(Ii) A host is transformed with the obtained recombinant phage or plasmid, and after culture, contains the target DNA by hybridization with a DNA probe encoding a part of the target protein or by immunoassay using an antibody. Isolate phage or plasmid.
(Iii) It can be produced by cutting out the desired cloned DNA from the recombinant DNA and ligating the cloned DNA or a part thereof downstream of the promoter in the expression vector.
Thereafter, the host is transformed with the expression vector and cultured by an appropriate method. Culture is usually performed at 15 to 43 ° C. for 3 to 24 hours, and aeration and agitation can be added as necessary.

  As the lysis method in the lysis process, any of physical disruption by ultrasound, treatment with a lytic enzyme such as lysozyme, treatment with a lysis agent such as a surfactant, etc. can be used. Preferably, from the standpoint that the useful protein is not denatured, a lysing agent such as a quaternary ammonium type cationic surfactant whose counter ion is a carboxylate ion such as formic acid or acetic acid can be mentioned.

Examples of the unfolding agent used in the unfolding step include guanidine hydrochloride, urea and a combination thereof.
In addition, when the protein is a protein containing an S—S bond in the molecule, 2-mercaptoethanol, dithiothreitol, cystine, thiophenol or the like is further added as a reducing agent in addition to guanidine hydrochloride and / or urea. May be.

  Examples of the packing material used for column chromatography in the protein separation / removal process include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer, etc., and commercially available products such as Sephadex series, Sephacryl series, Sepharose series (above, Pharmacia series). And Bio-Gel series (Bio-Rad) are available.

The protein of the present invention is a protein obtained by the above protein production method.
Examples of the protein obtained by the protein production method of the present invention include enzymes, recombinant proteins, and nucleic acids.

Examples of the enzyme include a hydrolase, an isomerase, an oxidoreductase, a transferase, a synthetic enzyme, and a desorbing enzyme.
Examples of hydrolases include protease, serine protease, amylase, lipase, cellulase, glucoamylase and the like.
An example of the isomerase is glucose isomerase.
Examples of the oxidoreductase include peroxidase.
Examples of the transferase include acyltransferase and sulfotransferase.
Synthetic enzymes include fatty acid synthase, phosphate synthase, citrate synthase and the like.
Examples of the desorption enzyme include pectin lyase.

Examples of the recombinant protein include protein preparations and vaccines.
Examples of protein preparations include interferon α, interferon β, interleukins 1 to 12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), natriuretic peptide, Examples include blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like.
Examples of the vaccine include hepatitis A vaccine, hepatitis B vaccine, and hepatitis C vaccine.

  Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

<Example>
The following examples further illustrate the invention, but the invention is not limited thereto. Refolding was performed by the methods of Examples 1 to 10 and Comparative Examples 1 to 9 below to obtain proteins. The enzyme activity of the obtained protein was measured.

Example 1
In a 10 ml sterilized test tube, 10 mg of lipase (“Lilipase” manufactured by Nagase ChemteX Corporation: the same shall be used hereinafter) and 6 mol / L guanidine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd .: hereinafter, the same shall be used) ) 1 ml of an aqueous solution was added and left at room temperature overnight to unfold the lipase. To this unfolded protein solution, 1.2 mol / L phosphate (A-1) (monosodium phosphate disodium: sodium dihydrogen phosphate = 1: 1 molar ratio) (both manufactured by Wako Pure Chemical Industries, Ltd .: Thereafter, 5 ml of the same was used, and the mixture was allowed to stand overnight at room temperature for refolding.
(Concentration of compound (A) in the system = 1.0 mol / L, concentration of protein in the refolding step in the system = 1.7 mg / mL)

Example 2
The same method as in Example 1 except that 1.2 mol / L sodium tripolyphosphate (A-2) (manufactured by Wako Pure Chemical Industries) was added instead of 1.2 mol / L phosphate (A-1). Refolded. (Concentration of compound (A) in the system = 1.0 mol / L, concentration of protein in the refolding step in the system = 1.7 mg / mL)

Example 3
A method similar to Example 1 except that 1.2 mol / L adenosine monophosphate sodium salt (A-3) (manufactured by SIGMA) is added instead of 1.2 mol / L phosphate (A-1). Refolded. (Concentration of compound (A) in the system = 1.0 mol / L, concentration of protein in the refolding step in the system = 1.7 mg / mL)

Example 4
The same method as in Example 1 except that 1.2 mol / L adenosine triphosphate sodium (A-4) (manufactured by SIGMA) is added instead of 1.2 mol / L phosphate (A-1). Refolded. (Concentration of compound (A) in the system = 1.0 mol / L, concentration of protein in the refolding step in the system = 1.7 mg / mL)

Example 5
Refolding was performed in the same manner as in Example 1 except that the concentration of 1.2 mol / L was changed to 5 mL of 0.6 mol / L phosphate (A-1).
(Concentration of compound (A) in the system = 0.5 mol / L, protein concentration in the refolding step in the system = 1.7 mg / mL)

Example 6
Refolding was performed in the same manner as in Example 1 except that the concentration was 5 mL of 6 mol / L phosphate (A-1). (Concentration of compound (A) in the system = 5 mol / L, concentration of protein in the refolding step in the system = 1.7 mg / mL)

Example 7
Refolding was performed in the same manner as in Example 1 except that 5 mol of 0.3 mol / L sodium tripolyphosphate was used instead of 1.2 mol / L phosphate (A-1). (Concentration of compound (A) in the system = 0.25 mol / L, protein concentration in the refolding step in the system = 1.7 mg / mL)

Example 8
Refolding was performed in the same manner as in Example 1 except that the amount of lipase was 1.7 mg instead of 10 mg. (Concentration of compound (A) in the system = 1.0 mol / L, concentration of protein in the refolding step in the system = 0.28 mg / mL)

Example 9
Refolding was performed in the same manner as in Example 1 except that the amount of lipase was 27 mg instead of 10 mg. (Concentration of compound (A) in the system = 1.0 mol / L, concentration of protein in the refolding step in the system = 4.5 mg / mL)

Comparative Example 1
[Refolding when the concentration of the compound (A) in the system is low]
Refolding was performed in the same manner as in Example 1 except that 5 mL of 0.22 mol / L phosphate (A-1) was used instead of 1.2 mol / L. (Concentration of compound (A) in the system = 0.18 mol / L, protein concentration in the refolding step in the system = 1.7 mg / mL)

Comparative Example 2
[Refolding when the concentration of the compound (A) in the system is too high]
Refolding was performed in the same manner as in Example 1 except that 5 mL of 7.4 mol / L phosphate (A-1) was used instead of 1.2 mol / L. (Concentration of compound (A) in the system = 6.2 mol / L, concentration of protein in the refolding step in the system = 1.7 mg / mL)

Comparative Example 3
[Refolding when concentrations of compound (A) and protein in the system are too low]
Instead of 1.2 mol / L, the concentration was 0.22 mol / L phosphate (A-1) 5 mL, and the amount of lipase was 1.1 mg, as in Example 1. Refolded. (Concentration of compound (A) in the system = 0.18 mol / L, concentration of protein in the refolding step in the system = 0.18 mg / mL)

Comparative Example 4
[Refolding when the concentration of the compound (A) in the system is low and the concentration of the protein in the system is too high]
Refolding in the same manner as in Example 1 except that 5 mL of 0.22 mol / L phosphate (A-1) was used instead of 1.2 mol / L, and the amount of lipase was 192 mg. Went. (Concentration of compound (A) in the system = 0.18 mol / L, concentration of protein in the refolding step in the system = 32.0 mg / mL)

Comparative Example 5
[Refolding at a concentration using a phosphorus buffer for pH adjustment]
Instead of 1.2 mol / L phosphate (A-1), 5 ml of a commercially available “1/15 mol / L phosphate buffer (pH = 7) aqueous solution” (manufactured by Wako Pure Chemicals, sodium phosphate buffer) Refolding was performed in the same manner as in Example 1 except that. (Concentration of compound (A) in the system = 0.05 mol / L, protein concentration in the refolding step in the system = 1.7 mg / mL)

Comparative Example 6
[Refolding method using phosphorus buffer for the purpose of pH adjustment and greatly diluted]
Since the enzyme activity was low in Comparative Example 5, a method of reducing the concentration of the denaturant by diluting was employed to improve the enzyme activity.
1 ml of 10 mg of lipase “lipase” and 6 mol / L guanidine hydrochloride aqueous solution was added to a 10 ml sterilized test tube, and allowed to stand overnight at room temperature to fully unfold the lipase.
Add 1 ml of the above enzyme solution and 200 ml of a 1/15 mol / L phosphate buffer (pH = 7) aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.) to a 210 ml sterilized bottle, stir lightly, and leave it overnight at room temperature for refolding. (Concentration of compound (A) in the system: 0.06 mol / L, protein concentration in refolding step: 0.05 mg / mL).

Comparative Example 7
[Refolding by dilution method-1]
To a 10 ml sterilized test tube, 10 ml of lipase “lipase” and 1 ml of 6 mol / L guanidine hydrochloride aqueous solution were added, and left overnight at room temperature to fully unfold the lipase. In a 210 ml sterilized bottle, 1 ml of the above enzyme solution and 200 ml of 0.1 mol / L Tris buffer (pH = 7) were stirred gently and left at room temperature overnight.
(Refolding step protein concentration = 0.05 mg / mL)

Comparative Example 8
[Refolding by dilution method-2]
To a 10 ml sterilized test tube, 1 ml of 10 mg of lipase “lipase” and 6M aqueous guanidine hydrochloride was added and left at room temperature overnight to fully unfold the lipase. Add 1 ml of the above enzyme solution and 70 ml of 0.05% cetyltrimethylammonium bromide (CTAB) (manufactured by Tokyo Kasei) to a 140 ml sterile bottle and leave it at room temperature for 1 hour. 2% cycloamylose (manufactured by Ezaki Glico) 30 ml of the solution was added and left overnight. (Refolding step protein concentration = 0.1 mg / mL)

Comparative Example 9
[Refolding by dialysis]
To a 10 ml sterilized test tube, 10 ml of lipase “lipase” and 1 ml of 6 mol / L guanidine hydrochloride aqueous solution were added, and left overnight at room temperature to fully unfold the lipase. 1 ml of the above enzyme solution was put into a dialysis machine, 0.1 mol / L Tris buffer (pH = 7) was gradually added, and diluted gradually to perform refolding. The procedure was terminated when 200 ml of Tris buffer was added.
(Refolding step protein concentration = 0.05 mg / mL)

<Measurement of enzyme activity>
To a 10 ml sterilized test tube, add 2 ml of 0.1 mol / L Tris buffer (pH = 7) and the protein solution obtained in Examples 1 to 9 or Comparative Examples 1 to 9 with a 5 μl micropipette and mix gently. It was. Furthermore, 1 ml of 3.5 mmol / Lp-nitrophenyl acetate solution was added, and the absorbance (400 nm) of the hydrolysis product p-nitrophenol was measured for 3 minutes with an ultraviolet-visible spectrophotometer (manufactured by Shimadzu Corporation, UV-2550). Each measurement was performed for 12 minutes, and the initial rate of hydrolysis reaction was calculated from the rate of increase in absorbance with respect to time.
Moreover, natural lipase aqueous solution of the same concentration as each protein concentration of Examples 1-9 or Comparative Examples 1-9 was prepared, and the initial rate of the hydrolysis reaction of p-nitrophenyl acetate was used in the same manner as described above. Calculated.
The enzyme activities (%) of Examples 1 to 9 and Comparative Examples 1 to 9 were calculated by the following formula.
Enzyme activity (%) = [initial rate when using the proteins obtained in Examples and Comparative Examples / initial rate when using natural lipase] × 100

<Evaluation of protein productivity>
Protein productivity was defined and evaluated with the following indicators. The results are shown in Tables 1 and 2.
Productivity index = protein concentration in the refolding process (mg / mL) x enzyme activity (%)

By using the refolding agent and the refolding method of the present invention, various useful proteins can be produced with high purity and efficiency.
Examples of the protein obtained by the protein production method of the present invention include enzymes, recombinant proteins, and nucleic acids.

Claims (5)

  A method for refolding an unfolded protein, wherein the unfolded protein is composed of phosphate, pyrophosphate, polyphosphate, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and salts thereof. Wherein the concentration of the compound (A) in the system in the process is 0.5 to 6 mol / L. Folding method. In the step of treatment with refolding agent, refolding method according to claim 1, wherein the use further surfactant (D). The refolding method according to claim 1 or 2 , wherein a pH adjuster (E) and / or a protein stabilizer (F) is further used in the step of treating with the refolding agent. The protein refolding method according to any one of claims 1 to 3 , wherein the protein concentration in the system in the step of treating with the refolding agent (C) is 0.2 to 30 mg / mL. Methods of producing proteins comprising the step of refolding the claims 1-4 refolding method of any.