JP2013146226A - Production method for membrane protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method capable of obtaining a large amount of membrane protein.SOLUTION: A production method for membranal protein which carries out the secretory production of membranal protein using the culture solution containing bacteria and surfactants (A) is preferably a production method using surfactants containing nonionic surfactants (A1) in which HLB is 0 to 8 as surfactants (A) and more preferably a production method using surfactants containing nonionic surfactants (A1) in which HLB is 0 to 8 and the following surfactant (A2) as surfactants (A). The surfactant (A2) is at least one sort selected from the group consisting of ampholytic surfactants (A2-1), anion surfactants (A2-2), cationic surfactants (A2-3), and nonionic surfactants (A2-4) in which HLB exceeds 8 and is 13 or less.

Description

  The present invention relates to a method for producing a membrane protein.

  Bacteria are widely used to produce amino acids and proteins. In particular, in recent years, genes of proteins that are useful in medicine and industry are introduced into bacteria using genetic engineering techniques, transformed, and the bacteria are cultured to express the protein, thereby efficiently producing the target protein. The technology to produce is known. Produced proteins are widely used as proteins for industrial use, food processing, pharmaceutical use and the like.

  Membrane protein is a protein present on the cell membrane, which tends to be an inactive inclusion body when it is produced by bacteria, and has a problem that the production amount is small (50 mg / L or less based on the volume of the culture solution). .

  In order to extract a membrane protein from bacteria in an active state, it is necessary to extract the bacterial membrane components together, and there is a problem that the process becomes complicated. For these reasons, membrane proteins have not been produced commercially and are not commercially used to date.

Journal of Biotechnology, Norio Kayama et al., 2000, Vol. 78, No. 3, p82-93 Valerie L THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, Vol. 274, no. 7, p4246-4253

  An object of this invention is to provide the production method which can obtain a membrane protein in large quantities.

As a result of intensive studies to solve the above problems, the present inventors have reached the present invention.
That is, the membrane protein production method of the present invention is a membrane protein production method in which a membrane protein is secreted and produced using a culture solution containing bacteria and a surfactant (A).

  The membrane protein production method of the present invention can obtain a large amount of membrane protein.

A membrane protein is a protein that exists in a lipid membrane of a cell and is a protein that binds to the lipid membrane of a cell in bacteria.
Membrane proteins are difficult to produce in large quantities, but can be obtained in large quantities according to the production method of the present invention. Furthermore, membrane proteins tend to be inactive compared to proteins other than membrane proteins, and it is difficult to obtain active proteins. However, according to the production method of the present invention, membrane proteins can be obtained in an active state. Is possible.
The membrane protein includes a conventionally known membrane protein, and is not particularly limited. Specifically, an enzyme {hyaluronic acid synthase (such as Streptococcus pyogenes or algal virus-derived hyaluronic acid synthase) and a reductase ( P450 etc.)} and antibodies (IgD etc.).

  Examples of the bacterium in the present invention are shown below, but are not limited thereto. Bacteria include eubacteria and archaea. Eubacteria include gram negative bacteria and gram positive bacteria. Gram-negative bacteria include Escherichia, Thermus, Rhizobium, Pseudomonas, Shewanella, Vibromo, Vibromo, Vibromo Examples include Salmonella, Acetobacter, Synechocystis, and the like. Examples of Gram-positive bacteria include Bacillus, Streptomyces, Corynebacterium, Brevibacterium, Bifidobacterium, Lactococcus cc. And bacteria included in the genus Enterococcus, Pediococcus, Leuconostoc, Streptomyces, and the like.

  Of these, from the viewpoint of producing a large amount of membrane protein, Gram-negative bacteria are preferable, Escherichia is more preferable, and Escherichia coli is more preferable.

The surfactant (A) used in the production method of the present invention includes a nonionic surfactant (A1) having an HLB of 0 to 8 and the following surfactant (A2).
Surfactant (A2): Amphoteric surfactant (A2-1), anionic surfactant (A2-2), cationic surfactant (A2-3), and nonionic property with HLB of more than 8 and 13 or less At least one selected from the group consisting of a surfactant (A2-4).

(A1) is an aliphatic monoalcohol alkylene oxide (hereinafter, abbreviated as AO) adduct (A1-1), alkylphenol AO adduct (A1-2), which is a surfactant having an HLB of 0 to 8. ), Fatty acid AO adduct (A1-3) and polyhydric alcohol type nonionic surfactant (A1-4).
HLB is known as a scale indicating the hydrophilicity and hydrophobicity of a nonionic surfactant, and the higher the HLB value, the higher the hydrophilicity. The HLB in the present invention is a numerical value calculated by the following formula (1) (Takehiko Fujimoto, Introduction to Surfactant, page 142, issued by Sanyo Chemical Industries, Ltd.).

HLB = 20 × {molecular weight of hydrophilic group / molecular weight of surfactant} (1)

  The HLB of (A1) is preferably more than 0 and 8 or less, more preferably more than 0 and 7 or less, and still more preferably 0.5 to 6, from the viewpoint of membrane protein stabilization. Especially preferably, it is 1-5.

  In the aliphatic monoalcohol AO adduct (A1-1), the aliphatic monoalcohol includes an aliphatic monoalcohol having 8 to 24 carbon atoms. Specific examples of the aliphatic monoalcohol include capryl alcohol, decyl alcohol, dodecyl alcohol, octadecyl alcohol, oleyl alcohol, myristyl alcohol, stearyl alcohol, cetyl alcohol, and coconut oil alkyl alcohol. As AO, C2-C4 AO is contained. Specific examples of AO include ethylene oxide (hereinafter referred to as EO) and propylene oxide (hereinafter referred to as PO). AO may use 2 or more types together. The number of added moles of AO includes 1 to 20 added moles when one kind of AO is used, and 2 to 40 added moles when two or more kinds of AO are used in combination. When AO is EO, the number of EO addition moles is decreased, and the HLB is lowered when the number of carbon atoms of the aliphatic alcohol is increased. Specific examples of (A1-1) include oleyl alcohol EO adduct and decyl alcohol EOPO adduct, etc., and oleyl alcohol EO2 mol adduct (HLB = 6.0) and EO8 mol PO7 mol block of decyl alcohol. An adduct (HLB8.0) etc. are mentioned.

  In the alkylphenol AO adduct (A1-2), the alkylphenol includes an alkylphenol having 7 to 30 carbon atoms, and examples thereof include butylphenol, pentylphenol, hexylphenol, octylphenol, nonylphenol, decylphenol, and dodecylphenol. As AO, C2-C4 AO is contained. Specific examples of AO include EO and PO. AO may use 2 or more types together. The number of added moles of AO includes 1 to 20 added moles when one kind of AO is used, and 2 to 40 added moles when two or more kinds of AO are used in combination. When AO is EO, HLB becomes small when the EO addition mole number is decreased. Specific examples of (A1-2) include nonylphenol EO adducts and the like, and nonylphenol EO2 molar adduct (HLB = 5.7) and the like.

  The fatty acid AO adduct (A1-3) is an esterified product of a fatty acid and a copolymer of AO. Examples of the fatty acid include fatty acids having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearin Acid, oleic acid, coconut oil fatty acid and the like). AO is AO having 2 to 4 carbon atoms, and examples thereof include EO and PO. AO may use 2 or more types together. The number of added moles of AO includes 1 to 20 added moles when one kind of AO is used, and 2 to 40 added moles when two or more kinds of AO are used in combination. When AO is EO, HLB becomes small when the EO addition mole number is decreased. Specific examples of (A1-3) include polyoxyethylene glycol dilaurate (oxyethylene polymerization degree = 5, HLB = 6.6), polyoxyethylene glycol dioleate (oxyethylene polymerization degree = 5, HLB = 5.6) and the like.

  Examples of the polyhydric alcohol type nonionic surfactant (A1-4) include EO and / or 2-8 polyhydric alcohols having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan). Or PO adducts; fatty acid esters of polyhydric alcohols and EO adducts thereof; fatty acid esters of sugars (monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides having 3 to 7 carbon atoms), fatty acid alkanolamides (coconut palm) Oil fatty acid diethanolamide and the like) and AO adducts thereof. Specific examples of (A1-4) include sorbitan trioleate (HLB = 1.8), sorbitan monooleate (HLB = 4.3), sorbitan monostearate (HLB = 4.7), and the like.

  In the production method of the present invention, by containing (A1) in the culture solution, since the membrane protein is secreted into the culture solution in a stabilized state, it can be taken out as an active membrane protein, A highly active membrane protein can be obtained as compared with a membrane protein extracted by crushing bacteria.

  Among (A1), from the viewpoint of membrane protein stabilization, alcohol AO adduct (A1-1), alkylphenol AO adduct (A1-2), fatty acid AO adduct (A1-3) having an HLB of 0 to 8 And a polyhydric alcohol type nonionic surfactant (A1-4), more preferably a polyhydric alcohol type nonionic surfactant (A1-4), particularly preferably sorbitan trioleate and sorbitan monooleate. And sorbitan monostearate.

  The amphoteric surfactant (A2-1) includes a carboxylate amphoteric surfactant (A2-1-1), a sulfate ester amphoteric surfactant (A2-1-2), and a sulfonate amphoteric interface. An activator (A2-1-3), a phosphate ester type amphoteric surfactant (A2-1-4) and the like are included.

  As the carboxylate-type amphoteric surfactant (A2-1-1), an amino acid-type amphoteric surfactant (A2-1-1-1), a betaine-type amphoteric surfactant (A2-1-1-2) and And imidazoline type amphoteric surfactant (A2-1-1-3).

The amino acid type amphoteric surfactant (A2-1-1-1) is an amphoteric surfactant having an amino group and a carboxyl group in the molecule, and examples thereof include compounds represented by the following general formula (1).
[R—NH— (CH ₂ ) _n —COO ⁻ ] _m M (1)
In general formula (1), R is a C1-C20 monovalent hydrocarbon group. n is an integer of 1 or more. m is an integer of 1 or more. M is a proton; or a monovalent or divalent cation such as alkali metal, alkaline earth metal, ammonium (including cations derived from amines and alkanolamines) and quaternary ammonium.
Further, as (A2-1-1-1), specifically, an alkylaminopropionic acid type amphoteric surfactant (sodium cocaminopropionate, sodium stearylaminopropionate, sodium laurylaminopropionate, etc.); alkylaminoacetic acid Type amphoteric surfactant (such as sodium laurylaminoacetate) and sodium N-lauroyl-N′-carboxymethyl-N′-hydroxyethylethylenediamine.

The betaine-type amphoteric surfactant (A2-1-1-2) is an amphoteric surfactant having a quaternary ammonium salt-type cation moiety and a carboxylic acid-type anion moiety in the molecule. Examples of (A2-1-1-2) include compounds represented by the following general formula (2). Specific examples of (A1-1-2) include alkyldimethylbetaine (such as stearyldimethylaminoacetic acid betaine and lauryldimethylaminoacetic acid betaine), amide betaine (such as coconut oil fatty acid amidopropylbetaine) (coconut oil fatty acid amidopropyldimethylaminoacetic acid). Betaine, hydrogenated coconut oil fatty acid amidopropyl dimethylaminoacetic acid betaine and the like) and lauric acid amidopropyl betaine and the like, and alkyldihydroxyalkyl betaines (lauryl dihydroxyethyl betaine and the like).
RN ⁺ (CH ₃ ) ₂ —CH ₂ COO ⁻ (2)
In general formula (2), R is a C1-C20 monovalent hydrocarbon group.

  Examples of the imidazoline-type amphoteric surfactant (A2-1-1-3) include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine.

  Other amphoteric surfactants include glycine-type amphoteric surfactants such as sodium lauroyl glycine, sodium lauryl diaminoethyl glycine, lauryl diaminoethyl glycine hydrochloride and dioctyl diaminoethyl glycine hydrochloride; sulfobetaine types such as pentadecyl sulfotaurine Amphoteric surfactants; Coleamidopropyldimethylammoniopropanesulfonic acid (CHAPS), Coleamidopropyldimethylammonio 2-hydroxypropanesulfonic acid (CHAPSO); Alkylamine oxide amphoteric surfactants such as lauryldimethylamine oxide included.

  As an anionic surfactant (A2-2), ether carboxylic acid (A2-2-1) and its salt, sulfate ester (A2-2-2) or its salt, ether sulfate ester (A2-2-3) And its salts, sulfonate (A2-2-4), sulfosuccinate (A2-2-5), phosphate ester (A2-2-6) and its salt, ether phosphate ester (A2-2-7) ) And salts thereof, fatty acid salts (A2-2-8), acylated amino acid salts, and naturally-occurring carboxylic acids and salts thereof (such as chenodeoxycholic acid, cholic acid, and deoxycholic acid).

  The ether carboxylic acid (A2-2-1) or a salt thereof includes an ether carboxylic acid having a hydrocarbon group (carbon number 8 to 24) and a salt thereof. Specific examples of (A2-2-1) or a salt thereof include polyoxyethylene lauryl ether acetic acid, polyoxyethylene lauryl ether acetate sodium salt, polyoxyethylene tridecyl ether acetate sodium salt, polyoxyethylene octyl ether acetate sodium salt And lauryl glycol acetate sodium salt.

  The sulfate ester (A2-2-2) and a salt thereof include a sulfate ester having a hydrocarbon group (8 to 24 carbon atoms) and a salt thereof. Specific examples of (A2-2-2) and its salts include sodium lauryl sulfate and triethanolamine lauryl sulfate.

  The ether sulfate ester (A2-2-3) and salts thereof include ether sulfate esters having a hydrocarbon group (8 to 24 carbon atoms) and salts thereof. Specific examples of (A2-2-3) and salts thereof include polyoxyethylene lauryl ether sulfate sodium salt and polyoxyethylene lauryl ether sulfate triethanolamine salt.

  Examples of the sulfonate (A2-2-4) include sodium dodecyl diphenyl ether disulfonate, sodium dodecylbenzenesulfonate, and sodium naphthalenesulfonate.

  Examples of the sulfosuccinate (A2-2-5) include polyoxyethylene lauryl sulfosuccinic acid disodium salt, sulfosuccinic acid lauryl disodium salt, and sulfosuccinic acid polyoxyethylene lauroylethanolamide disodium salt.

  Examples of the phosphate ester (A2-2-6) include octyl phosphate disodium salt and lauryl phosphate disodium salt.

  Examples of the ether phosphate (A2-2-7) include polyoxyethylene octyl ether phosphate disodium salt and polyoxyethylene lauryl ether phosphate disodium salt.

  Examples of the fatty acid salt (A2-2-8) include octylic acid sodium salt, lauric acid sodium salt, and stearic acid sodium salt.

  Examples of the cationic surfactant (A2-3) include an amine salt type cationic surfactant (A2-3-1) and a quaternary ammonium salt type cationic surfactant (A2-3-2). It is.

  As the amine salt type cationic surfactant (A2-3-1), primary to tertiary amines may be inorganic acids (hydrochloric acid, nitric acid, sulfuric acid, hydroiodic acid, etc.) or organic acids (acetic acid, formic acid, oxalic acid, lactic acid). , Gluconic acid, adipic acid, alkylphosphoric acid, etc.). For example, the primary amine salt type includes inorganic or organic acid salts of higher aliphatic amines (higher amines such as laurylamine, stearylamine, cetylamine, hardened tallow amine, and rosinamine); Examples include fatty acid (stearic acid, oleic acid, etc.) salts and the like. Examples of the secondary amine salt type include inorganic acid salts or organic acid salts such as ethylene oxide adducts of aliphatic amines. Examples of the tertiary amine salt type include aliphatic amines (triethylamine, ethyldimethylamine, N, N, N ′, N′-tetramethylethylenediamine, etc.), ethylene oxides of aliphatic amines (2 moles). Above) Adducts, alicyclic amines (N-methylpyrrolidine, N-methylpiperidine, N-methylhexamethyleneimine, N-methylmorpholine, 1,8-diazabicyclo (5,4,0) -7-undecene, etc.) , Inorganic acid salts or organic acid salts of nitrogen-containing heterocyclic aromatic amines (4-dimethylaminopyridine, N-methylimidazole, 4,4′-dipyridyl, etc.); triethanolamine monostearate, stearamide ethyl diethyl methyl ethanol Examples include inorganic acid salts or organic acid salts of tertiary amines such as amines.

  The quaternary ammonium salt type cationic surfactant (A2-3-2) includes tertiary amines and quaternizing agents (alkylating agents such as methyl chloride, methyl bromide, ethyl chloride, benzyl chloride, dimethyl sulfate, etc.) Those obtained by reaction with ethylene oxide, etc.). For example, lauryltrimethylammonium chloride, didecyldimethylammonium chloride, dioctyldimethylammonium bromide, stearyltrimethylammonium bromide, lauryldimethylbenzylammonium chloride (benzalkonium chloride), cetylpyridinium chloride, polyoxyethylenetrimethylammonium chloride, stearamide ethyl diethyl Examples include methylammonium methosulfate.

  Nonionic surfactants (A2-4) having an HLB of more than 8 and not more than 13 include aliphatic monoalcohol AO adducts (A2-4-1) and alkylphenol AO additions having an HLB of more than 8 and not more than 13. Product (A2-4-2), fatty acid AO adduct (A2-4-3), polyhydric alcohol type nonionic surfactant (A2-4-4) and the like.

  In the aliphatic monoalcohol AO adduct (A2-4-1), the aliphatic monoalcohol includes an aliphatic monoalcohol having 8 to 24 carbon atoms. Specific examples of the aliphatic monoalcohol include capryl alcohol, decyl alcohol, dodecyl alcohol, octadecyl alcohol, oleyl alcohol, myristyl alcohol, stearyl alcohol, cetyl alcohol, and coconut oil alkyl alcohol. As AO, C2-C4 AO is contained. Specific examples of AO include EO and PO. AO may use 2 or more types together. The number of added moles of AO includes 1 to 20 added moles when one kind of AO is used, and 2 to 40 added moles when two or more kinds of AO are used in combination. When AO is EO, the number of EO addition moles increases, and the HLB increases as the number of carbon atoms of the aliphatic alcohol decreases. Specific examples of (A2-4-1) include lauryl alcohol EO 7 mol adduct (HLB = 12.4), oleyl alcohol EO 5 mol adduct (HLB = 9.0), oleyl alcohol EO 6 mol adduct (HLB = 10.2), oleyl alcohol EO 7 mol adduct (HLB = 11.0), oleyl alcohol EO 10 mol adduct (HLB = 12.4), 1,2-dodecanediol monooxyethylene adduct (HLB = 10.4). ) And decyl alcohol EO 5 mol adduct (HLB = 10.3).

In the alkylphenol AO adduct (A2-4-2), the alkylphenol includes an alkylphenol having 7 to 30 carbon atoms, and examples thereof include butylphenol, pentylphenol, hexylphenol, octylphenol, nonylphenol, decylphenol, and dodecylphenol. It is done. As AO, C2-C4 AO is contained. Specific examples of AO include EO and PO. AO may use 2 or more types together. The number of added moles of AO includes 1 to 20 added moles when one kind of AO is used, and 2 to 40 added moles when two or more kinds of AO are used in combination. When AO is EO, the number of EO addition moles increases the HLB. Specific examples of (A2-4-2) include an EO1-20 mol and / or PO1-20 mol adduct of octylphenol and an EO1-20 mol and / or PO1-20 mol adduct of nonylphenol. Also, TRITON ^™ X-114 (HLB = 12.4), igepal ^™ CA-520 (HLB = 10.0), igepal ^™ CA-630 (HLB = 13.0) and the like are easily available from the market.

  The fatty acid AO adduct (A2-4-3) is an esterified product of a fatty acid and a copolymer of AO. As the fatty acid, a fatty acid having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid). , Stearic acid, oleic acid and coconut oil fatty acid). AO is AO having 2 to 4 carbon atoms, and examples thereof include EO and PO. AO may use 2 or more types together. The number of added moles of AO includes 1 to 20 added moles when one kind of AO is used, and 2 to 40 added moles when two or more kinds of AO are used in combination. When AO is EO, the number of EO addition moles increases the HLB. Specifically as (A2-4-3), oleic acid EO 9 mol adduct (HLB = 11.8), dioleic acid EO 12 mol adduct (HLB = 10.4), dioleic acid EO 20 mol adduct (HLB = 12.9) and stearic acid EO 9 mol adduct (HLB = 11.9).

  As the polyhydric alcohol type nonionic surfactant (A2-4-4), EO of a divalent to octavalent polyhydric alcohol having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan) is used. And / or PO adducts; fatty acid esters of polyhydric alcohols and EO adducts thereof, as well as fatty acid esters of sugar, fatty acid alkanolamides (such as coconut oil fatty acid diethanolamide) and their AO adducts. Specific examples of (A2-4-4) include sorbitan tetraoleate EO 32 mol adduct (HLB = 11.4) and sorbitan hexaoleate EO 46 mol adduct (HLB = 10.2). .

  In the production method of the present invention, by containing (A2) in the culture solution, the membrane protein is easily secreted into the culture solution, and a large amount of membrane protein can be obtained.

  Among (A2), from the viewpoint of secretion efficiency, amphoteric surfactants, anionic surfactants, and nonionic surfactants having an HLB of more than 8 and 13 or less are preferred, and carboxylate amphoteric surfactants are more preferred. (A2-1-1), ether carboxylic acid (A2-2-1), sulfonate (A2-2-4), aliphatic monoalcohol AO adduct (A2-4-1), and polyhydric alcohol type nonion Surfactant (A2-4-4), particularly preferably sodium polyoxyethylene tridecyl ether acetate (sodium salt of polyoxyethylene tridecyl ether acetic acid), coconut oil fatty acid diethanolamide, coconut oil fatty acid amidopropyl Dimethylaminoacetic acid betaine, 1,2-dodecanediol monooxyethylene adduct, lauryldimethylaminoacetic acid betaine , Hardened coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine, sodium cocaminopropionate, sodium dodecyldiphenyl ether disulfonate, sodium polyoxyethylene lauryl ether acetate (sodium salt of polyoxyethylene lauryl ether acetic acid), aliphatic monoalcohol EO Additives, polyoxyethylene lauryl ether sulfate sodium salt (sodium salt of polyoxyethylene lauryl ether sulfate) and decyl alcohol EO adduct.

In the production method of the present invention, the surfactant (A) may be used singly or in combination of two or more.
Moreover, when using 2 or more types, it is preferable to use together (A1) and (A2) from a viewpoint of secretion efficiency and membrane protein stabilization, More preferably, the polyhydric alcohol type nonionic property of HLB 0-8 A combination of a surfactant (A1-4) and a betaine-type amphoteric surfactant (A2-1-1-2), particularly preferably a combination of sorbitan monooleate and coconut oil fatty acid amide propylbetaine.
By using (A1) and (A2) in combination, a more active membrane protein can be produced in a larger amount.

In the present invention, as the surfactant (A), the surfactant (A) may be used as it is, or may be mixed with water as necessary and used as an aqueous diluent (aqueous solution or aqueous dispersion). .
The total concentration of the surfactant (A) in the aqueous dilution is appropriately selected depending on the target bacteria, the type of membrane protein, and the type of extraction method. From the viewpoint of membrane protein production and handling properties, 0.1 to 99% by weight is preferred, preferably 1 to 50% by weight, based on the weight of the diluent.

  From the viewpoint of productivity, the secretion efficiency (%) when membrane protein is produced using a surfactant is preferably 1 to 100, more preferably 5 to 100, and even more preferably 10 to 100, Especially preferably, it is 50-100.

The secretion efficiency of the surfactant indicates that the membrane protein in the bacteria is secreted outside the bacteria (in the culture solution) by the surfactant.
In the present invention, it is defined by the following formula.
Secretion efficiency (%) = 100 × {(X / Y) −Z}
X: Membrane protein in the culture solution remaining after removal of the cells by centrifugation Y: Total membrane protein in the culture solution Z: The ratio of the lysed bacteria, defined by the following formula.
Z = Z1 / Z2
Z1: Localized substance in the cytoplasm in the culture solution remaining after removal of the cells by centrifugation Z2: Localized substance in the cytoplasm in the cultured solution Note that the localized substance in the cytoplasm is a substance present in the cytoplasm This refers to substances that are eluted into the culture medium by lysis.

  For example, if the membrane protein produced in bacteria moves more to the outer membrane, the secretion efficiency increases, and if the membrane protein does not move more to the outer membrane, the secretion efficiency decreases. Moreover, secretion efficiency can be raised by selecting surfactant with high secretion efficiency by screening.

In the production method of the present invention, when only the surfactant (A1) is contained, the content (% by weight) of (A1) in the culture solution depends on the target bacteria, the type of membrane protein produced and the extraction method. Although it is appropriately selected depending on the type, from the viewpoint of obtaining membrane protein productivity and highly active membrane protein based on the weight of the culture solution, 0.01 to 15 is preferable, more preferably 0.1 to 10, particularly Preferably it is 0.1-5.
In addition, in the production method of the present invention, when only the surfactant (A2) is contained, the content (% by weight) of (A2) in the culture solution depends on the target bacteria, the type of membrane protein produced and Although it is appropriately selected depending on the type of extraction method, 0.01 to 15 is preferable, and more preferably 0.1 to 15 from the viewpoint of obtaining membrane protein productivity and highly active membrane protein based on the weight of the culture solution. 10, particularly preferably 0.1 to 5.

When the surfactant (A1) and the surfactant (A2) are used in the production method of the present invention, the content of the surfactant (A) in the culture broth (% by weight, the surfactant (A1) and the interface) The total amount of the active agent (A2)) is appropriately selected depending on the target bacteria, the type of membrane protein to be produced and the type of extraction method. In light of obtaining an active membrane protein, 0.01 to 15 is preferable, 0.1 to 10 is more preferable, and 0.1 to 5 is still more preferable.
In addition, when the surfactant (A1) and the surfactant (A2) are used in the production method of the present invention, the content (% by weight) of (A1) in the culture solution is the target bacteria. However, 0.01 to 15 is preferable from the viewpoint of obtaining membrane protein productivity and highly active membrane protein based on the weight of the culture solution. Preferably it is 0.1-10, Most preferably, it is 0.1-5.
In addition, when the surfactant (A1) and the surfactant (A2) are used in the production method of the present invention, the content (% by weight) of (A2) in the culture solution is the target bacteria produced. However, 0.01 to 15 is preferable from the viewpoint of obtaining membrane protein productivity and highly active membrane protein based on the weight of the culture solution. Preferably it is 0.1-10, Most preferably, it is 0.1-5.
In the production method of the present invention, when the surfactant (A1) and the surfactant (A2) are used, the weight ratio ((A1) / (A2)) of (A1) and (A2) in the culture solution is From the viewpoint of secretion efficiency and membrane protein stabilization, 0.1 to 10 is preferable, more preferably 0.5 to 5, and particularly preferably 1 to 2.

Surfactant (A) may be added later to a culture solution in which bacteria are suspended in a medium, in addition to being used in advance mixed with a medium. When added later, from the viewpoint of obtaining a large amount of membrane protein, it is preferable to add the surfactant (A) 3 to 72 hours after the cultivation.
Mixing with a culture medium can be performed by adding surfactant (A) to a culture solution at 4 to 99 ° C. and stirring with a stirring blade or a stirrer. When mixing afterwards, it can carry out by adding, stirring with a stirring blade etc.

The culture solution generally contains bacteria and a medium, and is a liquid for culturing bacteria. In the present invention, the culture solution further contains a surfactant (A).
The medium refers to a medium that provides a growth environment in culturing microorganisms, and specifically refers to a medium in which a carbon source, a nitrogen source, inorganic salts, and the like are dissolved in an aqueous medium such as water. The medium only needs to contain a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism. The amount of each component is not limited and can be appropriately selected by those skilled in the art. Specific examples of the culture medium include gravy medium, gravy gelatin medium, nitrate medium, milk medium, corn meal medium, MR-VP medium, tomato juice medium, TSI medium, SIM medium, glucose-bouillon medium, YM medium, and bacteria. Complete medium, Lennox medium (L medium), LB medium, minimal medium for E. coli (Davis medium), minimal salt medium for E. coli (MS), Tris-glucose medium (TG medium), EMB sugar indicator medium, minimal medium for Bacillus subtilis (Spizizen minimum medium), inorganic salt solution, Bacillus subtilis minimum salt medium, Bacillus subtilis transformation medium I, Bacillus subtilis transformation medium II, yeast complete medium (YPAD), yeast minimal medium, red bread mold complete medium , Minimum medium for red mold (Vogel medium N), minimum medium for red mold fungus, complete medium for Aspergillus (ANA medium) And Czapek - Dogs medium, and the like.
Moreover, you may melt | dissolve a carbon source, a nitrogen source, inorganic salts, etc. further in a culture solution as needed. As the carbon source, bacteria can synthesize ATP (adenosine triphosphate) and can be assimilated by metabolism, and include sugars such as glucose, lipids and proteins. Nitrogen sources are those that bacteria can use for the biosynthesis of proteins and nucleic acids, and include proteins, amino acids, ammonia, urea, and the like. Examples of inorganic salts include ammonium sulfate.
The amount of carbon source, nitrogen source, inorganic salts, etc. dissolved in the culture solution is not particularly limited.

In the present invention, the dry cell density represents the weight of bacteria contained in 1 L of the culture solution at any time from the start of culture to the end of culture in secretory production of membrane proteins. The weight of the bacteria is the weight of the dried bacteria.
The dry cell density is determined by the following procedures (1) to (5).
Procedure (1): The weight of the container (centrifugal tube) is measured in advance.
Procedure (2): 100 ml of the culture solution is placed in the container whose weight was measured in Procedure (1), centrifuged (4,000 G, 15 minutes, 4 ° C.), the supernatant is extracted, and the bacteria are collected.
Procedure (3): The collected bacteria in the container are washed with 0.9% by weight NaCl aqueous solution [the same volume as the culture solution used in Procedure (2)] and centrifuged again (4,000 G, 15 minutes, 4 ℃), remove the supernatant and collect the bacteria.
Procedure (4): The bacteria obtained in the procedure (3) are dried in a container at 105 ° C. for 10 hours, and the total weight of the container and the bacteria is measured.
Procedure (5): After procedure (4), after further drying at 105 ° C. for 2 hours, the total weight of the container and bacteria is measured to confirm that there is no change in weight. If the weight further decreases, drying is continued at 105 ° C. until there is no change in weight.
The dry cell density is determined by applying the measured value of procedure (5) and procedure (1) and the volume (L) of the culture solution used in procedure (2) to the following equation.
Dry cell density (g / L) = ([measured value of procedure (5)] − [measured value of procedure (1)]) / 0.1

The dry cell density in the production method of the present invention is preferably 1.5 to 500 g / L, more preferably 3 to 200 g / L, and particularly preferably 4 to 100 g / L based on the volume of the culture solution. . When the dry cell density is 1.5 g / L or more, the production amount of the membrane protein becomes high, and when it is 500 g / L or less, the bacterial protein can be efficiently secreted and produced without dying bacteria. it can.
The dry cell density when the bacterium is Escherichia coli is preferably 1.5 to 500 g / L, more preferably 3 to 100 g / L, from the viewpoint that membrane protein can be produced based on the volume of the culture solution. And then more preferably 10 to 50 g / L, most preferably 12 to 27 g / L.
In the membrane protein production method of the present invention, the amount of membrane protein production increases as the dry cell density increases within the above range.

  In the method for producing a membrane protein of the present invention, from the viewpoint of the production amount of the membrane protein, the time during which the dry cell density is within the above range is preferably 10% or more of the time required for the step of secreting the membrane protein. More preferably, it is 50% or more.

  Dry cell density can be increased, for example, by feeding at an appropriate rate using a semi-batch culture method under sufficient aeration conditions and reduced by performing batch culture under limited aeration conditions. Can do. Further, it can be increased by increasing the time from the start of culture to the addition of the surfactant, and can be decreased by shortening the time from the start of the culture to the addition of the surfactant.

In the method for producing a membrane protein of the present invention, the production method for secretory production of a membrane protein includes an extracellular secretion production method including the following steps (a) and (b). In the following steps, the step of producing secreted membrane protein is step (a).
Step (a): A step of causing a membrane protein to be secreted extracellularly (in a culture solution) by causing a bacterium (such as a Gram-negative bacterium) producing a membrane protein and a surfactant to be present simultaneously.
Step (b): A step of separating membrane protein from the culture solution after step (a).

An example of a method for producing a membrane protein using the surfactant of the present invention is shown below.
(I) Genetic recombination (i-1) Isolating messenger RNA (mRNA) from cells expressing the target protein, synthesizing single-stranded cDNA and then double-stranded DNA from the mRNA, Strand DNA is incorporated into phage DNA or plasmid. The resulting recombinant phage or plasmid is transformed into host E. coli to prepare a cDNA library.
(I-2) As a method for screening phage DNA or plasmid containing the target DNA, a hybridization method of the phage DNA or plasmid and a DNA probe encoding a part of the target membrane protein gene or complementary sequence Is mentioned.
(I-3) The target cloned DNA or a part thereof is cut out from the screened phage or plasmid, and the cloned DNA or a part thereof is ligated downstream of the promoter in the expression vector. Expression vectors can be prepared. DNAs encoding a signal sequence to be transferred to the outer membrane can be linked simultaneously.
(Ii) Culture (ii-1) A host bacterium is transformed with an expression vector to produce a recombinant bacterium, and the recombinant bacterium is pre-cultured. Pre-culture is usually performed at 15 to 43 ° C. for 3 to 72 hours on an agar medium.
(Ii-2) The culture solution used for the production of membrane protein is autoclaved at 121 ° C. for 20 minutes, and the recombinant bacteria pre-cultured on an agar medium are cultured here. The culture is usually performed at 15 to 43 ° C. for 12 to 72 hours. When the surfactant (A) is used simultaneously with the start of the culture, the pre-cultured recombinant bacteria are cultured in the same manner using a mixture of the surfactant (A) and the medium that has been homogenized. Moreover, when adding surfactant (A) 6 to 72 hours after culture | cultivation, after adding surfactant, culture | cultivation is continued for 1 to 1000 hours.
(Iii) Purification (iii-1) The protein secreted into the culture solution is separated from microorganisms and microbial residues by centrifugation, hollow fiber separation, filtration and the like.
(Iii-2) The culture solution containing membrane protein is subjected to precipitation treatment such as ethanol precipitation, ammonium sulfate precipitation and polyethylene glycol precipitation by repeating column treatments such as ion exchange column, gel filtration column, hydrophobic column, affinity column and ultra-column. Is separated and purified by appropriately performing as necessary.

  Thereafter, the host cell isolated in (iii-1) can be further cultured by supplying a new culture solution. By further subjecting the culture medium and the like to the step (iii) and repeating purification and culture, continuous production of membrane protein can be performed.

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

  By using the membrane protein production method of the present invention, a high yield can be obtained in a short time, and thus a high production amount can be achieved. Moreover, since the membrane protein is secreted into the culture solution, the membrane protein production method of the present invention facilitates purification of the membrane protein.

The method for producing a membrane protein of the present invention includes a step in which a surfactant and a bacterium are simultaneously present to secrete the membrane protein into the culture solution. In this step, as long as the bacteria are alive, it is presumed that the bacteria can make a membrane protein and secrete it into the culture medium. Furthermore, it is speculated that the production method of the present invention can be used regardless of the type of membrane protein to be produced if the bacteria have the ability to produce a membrane protein.
The membrane protein production method of the present invention is particularly effective when a membrane protein prepared in bacteria is transferred to the outer membrane of bacteria. When the membrane protein is transferred to the outer membrane, the membrane protein is easily secreted into the culture solution.

  The present invention will be further described with reference to the following examples and comparative examples, but the present invention is not limited thereto.

  Creation of strains and measurement of cell weight were performed based on standard methods performed by those skilled in the art.

<Production Example 1>
The NdeI restriction enzyme site and the XhoI restriction enzyme of the pET-22b plasmid (Novagen) containing the nucleic acid sequence (Invitrogen) in which the Escherichia coli-derived ADEA outer membrane translocation signal incorporated into the pMX vector and Streptococcus pyogenes-derived hyaluronic acid synthase are combined. Joined to the site. Then, using λDE3 Lysogenization Kit (Novagen), this plasmid was transformed into the AG1 (DE3) E. coli strain prepared by modifying E. coli strain AG1 (TOYOBO) to create a hyaluronic acid synthase expression strain (α) did.

<Measurement of amount of hyaluronic acid synthase>
Using the culture supernatant obtained in Example 1 and Comparative Example 1, the amount of hyaluronic acid synthase contained in the culture supernatant was measured. SDS-PAGE was performed for 1 μg, 2.5 μg, and 5 μg of bovine serum albumin as a calibration curve for the amount of protein. Proteins were stained with Coomassie Brilliant Blue stain. A calibration curve was prepared from the bovine serum albumin band. The amount of hyaluronic acid synthase contained in the culture supernatant was calculated using a calibration curve.

<Measurement of hyaluronic acid synthesis>
UDP-N-acetylglucosamine and UDP-glucuronic acid were added to 10 μL of the culture supernatant containing hyaluronic acid synthase obtained in Examples 1 to 4 and Comparative Example 1 so as to be 0.1% by weight, respectively. , Mixed in a buffer solution, and allowed to react at 30 ° C. for 2 hours.
After the reaction, the reaction solution was electrophoresed in a polyacrylamide gel. The electrophoresed gel was stained with Alcian blue staining solution. At the same time, 12.5 ng, 25 ng and 50 ng of purified hyaluronic acid were electrophoresed and stained. A calibration curve for hyaluronic acid was prepared from the band of hyaluronic acid observed during electrophoresis. Using the calibration curve, the amount of synthesized hyaluronic acid was calculated from the hyaluronic acid band observed during electrophoresis.

<Specific activity of hyaluronic acid synthase>
The specific activity of hyaluronic acid synthase was calculated by the following formula using the obtained hyaluronic acid synthase amount and hyaluronic acid synthesis value.
Specific activity = (hyaluronic acid synthesis amount) / (hyaluronic acid synthase amount)

<Comparative Example 1>
Prepare 1 ml of the overnight culture solution of E. coli (α) obtained in Production Example 1, inoculate 0.5 ml of LB medium (containing ampicillin 100 mg / L), shake culture at 30 ° C for 3 hours, and pre-culture A liquid was created. The pre-culture solution was 50 mL of medium (1.2 g yeast extract (manufactured by Nippon Pharmaceutical), 0.6 g polypeptone (manufactured by Nippon Pharmaceutical), 0.47 g dipotassium phosphate, 0.11 g monopotassium phosphate, ammonium sulfate 35 g, disodium phosphate dodecahydrate 0.66 g, sodium citrate dihydrate 0.02 g, glycerol 0.2 g, lactalbumin hydrolyzate 1.5 g, antifoam (manufactured by Shin-Etsu Silicone, “KM-70 ”) 0.3 g, 1 mM magnesium sulfate, trace metal solution (calcium chloride 18.9 μg, iron (III) chloride 500 μg, zinc sulfate heptahydrate 9.0 μg, copper sulfate 5.1 μg, manganese chloride tetrahydrate 6) 7 μg, cobalt chloride 4.9 μg, ethylenediaminetetraacetic acid tetrasodium 200 μg), 100 mg / L ampicillin) , Culture was started while maintaining the pH6.8,30 ℃ by using the product name "BioJr.8"). Three hours after the start of the culture, 0.15 mL of 1M IPTG (isopropyl-β-thiogalactopyranoside) was added. 14 hours after the start of culture, glycerin / protein solution (50% by weight glycerin, 50 g / L lactalbumin hydrolyzate, 33 g / L antifoam (manufactured by Shin-Etsu Silicone, “KM-70”), 100 mg / L ampicillin) was added dropwise. The culture was started.
After 36 hours from the start of the culture, the culture solution was collected and centrifuged (4,000 G, 15 minutes, 4 ° C.) to remove the cells and obtain a culture supernatant.
Using the obtained culture supernatant, the amount of hyaluronic acid synthase, the amount of hyaluronic acid synthesized, and the specific activity of hyaluronic acid synthase were measured. The results are shown in Table 1.

<Example 1>
The same procedure as in Comparative Example 1 was performed until the culture was started.
14 hours after the start of the cultivation, sorbitan monooleate (HLB = 4.3) as the surfactant (A1) and coconut oil fatty acid as the surfactant (A2) so as to be 1% by weight based on the weight of the culture solution, respectively. Amidopropyl betaine was added. After 36 hours from the start of the culture, the culture solution was collected and centrifuged (4,000 G, 15 minutes, 4 ° C.) to remove the cells and obtain a culture supernatant.
Using the obtained culture supernatant, the amount of hyaluronic acid synthase, the amount of hyaluronic acid synthesized, and the specific activity of hyaluronic acid synthase were measured. The results are shown in Table 1.

<Example 2>
The same procedure as in Comparative Example 1 was performed until the culture was started.
14 hours after the start of the cultivation, sorbitan trioleate (HLB = 1.8) as the surfactant (A1) and coconut oil fatty acid as the surfactant (A2) so as to be 1% by weight based on the weight of the culture solution, respectively. Amidopropyl betaine was added. After 36 hours from the start of the culture, the culture solution was collected and centrifuged (4,000 G, 15 minutes, 4 ° C.) to remove the cells and obtain a culture supernatant.
Using the obtained culture supernatant, the amount of hyaluronic acid synthase, the amount of hyaluronic acid synthesized, and the specific activity of hyaluronic acid synthase were measured. The results are shown in Table 1.

<Example 3>
The same procedure as in Comparative Example 1 was performed until the culture was started.
14 hours after the start of the culture, sorbitan monostearate (HLB = 4.7) as the surfactant (A1) and coconut oil fatty acid as the surfactant (A2) so as to be 1% by weight based on the weight of the culture solution, respectively. Amidopropyl betaine was added. After 36 hours from the start of the culture, the culture solution was collected and centrifuged (4,000 G, 15 minutes, 4 ° C.) to remove the cells and obtain a culture supernatant.
Using the obtained culture supernatant, the amount of hyaluronic acid synthase, the amount of hyaluronic acid synthesized, and the specific activity of hyaluronic acid synthase were measured. The results are shown in Table 1.

<Example 4>
The same procedure as in Comparative Example 1 was performed until the culture was started.
14 hours after the start of cultivation, EO8 mol PO7 mol block adduct of decyl alcohol (HLB8.0), surfactant (A2) as a surfactant (A1) so as to be 1% by weight based on the weight of the culture solution, respectively. As coconut oil fatty acid amidopropyl betaine was added. After 36 hours from the start of the culture, the culture solution was collected and centrifuged (4,000 G, 15 minutes, 4 ° C.) to remove the cells and obtain a culture supernatant.
Using the obtained culture supernatant, the amount of hyaluronic acid synthase, the amount of hyaluronic acid synthesized, and the specific activity of hyaluronic acid synthase were measured. The results are shown in Table 1.

<Example 5>
The same procedure as in Comparative Example 1 was performed until the culture was started.
14 hours after the start of culture, coconut oil fatty acid amidopropyl betaine was added as a surfactant (A2) so as to be 1% by weight based on the weight of the culture solution. After 36 hours from the start of the culture, the culture solution was collected and centrifuged (4,000 G, 15 minutes, 4 ° C.) to remove the cells and obtain a culture supernatant.
Using the obtained culture supernatant, the amount of hyaluronic acid synthase, the amount of hyaluronic acid synthesized, and the specific activity of hyaluronic acid synthase were measured. The results are shown in Table 1.

From the results of Table 1, it can be seen that in Comparative Example 1 in which the surfactant (A) is not used, the amount of hyaluronic acid synthase, which is a membrane protein contained in the culture supernatant, is as small as 10 mg / L. On the other hand, in Examples 1 to 5 cultured using the surfactant (A), the amount of hyaluronic acid synthase in the culture supernatant was extremely large at 165 mg / L or more, and by using the production method of the present invention, It can be seen that a large amount of membrane protein can be obtained.
Moreover, the hyaluronic acid synthase obtained in Examples 1-5 was much higher than the hyaluronic acid synthase obtained in Comparative Example 1 with a specific activity of 0.18 mg / mg / h or more. In particular, Examples 1 to 4 in which surfactants (A1) and (A2) are used in combination have higher specific activity than Example 5 in which only (A2) is used, and (A1) and (A2) are used in combination. Thus, it can be seen that a membrane protein having a higher specific activity can be obtained.
From the above, it can be seen that a large amount of membrane protein can be obtained by using the production method of the present invention, and the activity of the obtained membrane protein is high.

  The membrane protein production method of the present invention can obtain a large amount of membrane protein. The obtained membrane protein can be suitably used for enzyme reaction, food processing, detergent, fiber treatment, papermaking, enzyme conversion, and the like.

Claims (5)

A method for producing a membrane protein, wherein the membrane protein is secreted and produced using a culture solution containing bacteria and a surfactant (A). The method for producing a membrane protein according to claim 1, wherein the surfactant (A) comprises a nonionic surfactant (A1) having an HLB of 0 to 8. The method for producing a membrane protein according to claim 1 or 2, wherein the surfactant (A) comprises a nonionic surfactant (A1) having an HLB of 0 to 8 and the following surfactant (A2).
Surfactant (A2): Amphoteric surfactant (A2-1), anionic surfactant (A2-2), cationic surfactant (A2-3), and nonionic property with HLB of more than 8 and 13 or less At least one selected from the group consisting of a surfactant (A2-4). The method for producing a membrane protein according to any one of claims 1 to 3, wherein the content of the surfactant (A) in the culture solution is 0.1 to 10% by weight based on the weight of the culture solution. The method for producing a membrane protein according to any one of claims 2 to 4, wherein the content of the nonionic surfactant (A1) in the culture solution is 0.1 to 10% by weight based on the weight of the culture solution.