JP2010220539A - Bacterium deficient in spore formation ability and method for producing protein using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide Brevibacillus formosus deficient in spore formation ability, and a method for producing a genetically engineered protein using the bacterium. <P>SOLUTION: The mutant strain of the bacterium is deficient in spore formation ability and has reduced protease activity by treating a wild strain of Brevibacillus formosus with a mutagenic substance. The method for producing the protein using Brevibacillus formosus includes transforming an expression plasmid containing a DNA sequence encoding a human type Fc receptor FcγRI into the mutant strain. The method for producing a recombinant protein has a slight environmental impact while having high protein production ability. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a bacterium belonging to the genus Bacillus that is deficient in sporulation ability and a method for producing a recombinant protein, particularly an Fc receptor, using the same.

  Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Fc receptors are classified according to the type of immunoglobulin to which they bind, and include Fcγ receptors that bind to the Fc region of IgG, Fcε receptors that bind to the Fc region of IgE, Fcα receptors that bind to the Fc region of IgA, etc. Patent Document 1). Each receptor is further classified according to the difference in structure. In the case of an Fcγ receptor, the presence of FcγRI, FcγRII, and FcγRIII has been reported (Non-patent Document 1).

FcγRI, which is one of the Fcγ receptors, is expressed in monocytes and macrophages and is inducibly expressed by γ interferon in neutrophils (Non-patent Document 1). FcγRI has high binding affinity for IgG, and its equilibrium dissociation constant (Kd) is 10 ⁻⁸ M or less (Non-patent Document 2). FcγRI is divided into an extracellular region, a transmembrane region, and an intracytoplasmic region, and binding to IgG occurs in the Fc region of IgG and the extracellular region of FcγRI, and then a signal is transmitted to the cytoplasm. FcγRI is composed of two types of subunits, an α chain with a molecular weight of about 42000 that is directly involved in binding to IgG, and a γ chain. The γ chain covalently binds at the boundary between the cell membrane and the extracellular region, thereby forming a homodimer. (Non-Patent Document 3).

  The amino acid sequence of human FcγRI and the gene sequence (SEQ ID NO: 1) are published in public databases such as ExPASy (Primary accession number: P12314). In addition, the functional domain in the structure of FcγRI, the signal peptide sequence for penetrating the cell membrane, and the position of the transmembrane region are also published, and FIG. 1 shows a schematic diagram of the structure of human FcγRI. The amino acid numbers in the figure correspond to the amino acid numbers described in SEQ ID NO: 1. That is, from methionine (Met) of amino acid number 1 of SEQ ID NO: 1 to valine (Val) of 289, extracellular region, from leucine (Leu) of amino acid number 294 of SEQ ID NO: 1 to threonine (Thr) of 374 penetrates the cell membrane. Regions and intracellular regions.

  In recent years, the unexpected immunosuppressive biological properties of Fc receptors have attracted attention as pharmaceuticals, particularly in the areas of autoimmune disease or autoimmune syndrome, transplant rejection and malignant lymphoproliferation. Reference 2). Further, the antibody adsorption ability, which is a function of FcγRI, can also be used as a protein responsible for the capture function of various antibody purification chromatography gels.

  The amino acid sequence and gene base sequence of the FcγRIα chain (Non-patent Document 4) were clarified by Janet et al., And then expression using E. coli (Patent Document 1) or animal cells has been reported by genetic recombination techniques. However, in the expression system using E. coli, the expression amount of the extracellular region protein of FcγRI is extremely low, and the expressed protein is expressed in the cell, so that the expressed protein often becomes an insoluble inclusion body. Inclusion body protein can be prepared as an active protein by an operation such as solubilization, but it requires a complicated operation. Moreover, in the system using animal cells, an expression level higher than that of Escherichia coli has been reported (Non-patent Document 3), but it takes a long time to culture and the productivity is not high.

  Previously, the present applicant has obtained a Bacillus bacterium mutant having a reduced protease (proteolytic enzyme) activity by treatment with a mutagen, and uses the mutant as a host for genetically producing FcγRI. By using this, the degradation of the expressed protein derived from the protease possessed by the wild strain of the genus Bacillus is reduced, and as a result, FcγRI is efficiently produced (Japanese Patent Application No. 2009-008680). However, since the genetically modified mutant obtained by the above invention has the ability to survive in the natural environment, when the recombinant is leaked from a manufacturing facility or the like in the course of culture or disposal, the recombinant There was concern about the environmental impact.

  As a Bacillus genus mutant strain that does not have the ability to survive in a natural environment, there is a mutant strain that is deficient in sporulation ability, and Brevibacillus choshinensis SP3 strain (manufactured by Takara Bio Inc.) is known. Patent Document 2 discloses that a mutant strain deficient in spore-forming ability is used in culture and fermentation processes. However, there are no reports on mutant strains deficient in the spore-forming ability of Brevibacillusformus (NBRC15716) useful for protein production by genetic engineering techniques.

JP-T-2004-530419 JP 2006-527589 A

J. et al. V. Ravetch et al., Annu. Rev. Immunol. , 9, 457 (1991) Toshiyuki Takai, Jpn. J. et al. Clin. Immunol. , 28, 318 (2005) A. Paetz et al., Biochem. Biophys. Res. Commun. 338, 1811 (2005) J. et al. M.M. Allen et al., Science, 243, 378 (1989) David M.M. Hoover et al., Nucleic Acid Res. , 30, e43 (2002) Gang Wu et al., Protein Expr Purif. 47, 441-445 (2006)

  An object of the present invention is to provide a mutant strain lacking the spore-forming ability of Brevibacillus formus useful for protein production by a genetic engineering technique, and a method for producing a protein by genetic engineering using the mutant strain. is there.

The present invention made in view of the above problems includes the following inventions:
(1) A Brevibacillusformus mutant strain deficient in sporulation ability, obtained by treating a Brevibacillusformus wild strain (NBRC15716) with a mutagen.

  (2) The mutant strain according to (1), wherein the protease activity is further reduced as compared to a Brevibacterium formus wild strain (NBRC15716).

  (3) A Brevibacillus mutant mutant which can express the protein obtained by transforming an expression plasmid containing a DNA sequence encoding the protein into the mutant described in (1) or (2).

  (4) The recombinant according to (3), wherein the protein is a human Fc receptor FcγRI.

  (5) A method for producing a human Fc receptor FcγRI using the recombinant according to (4).

  The present invention is described in further detail below.

  In general, as a method for obtaining mutant strains, in addition to selecting superior strains derived from natural mutations, productivity can be achieved after accelerating mutations by treating cells with mutagens or ultraviolet rays. Examples thereof include a method of selecting improved strains and a method of cell fusion of strains having different properties obtained by the above method. Among these, the mutant strain of the present invention is characterized by being obtained by treating a Brevibacterium formosus wild strain (NBRC15716) with a mutagen and lacking the spore-forming ability.

  Examples of mutagens used for obtaining the Brevibacillus formusus mutant of the present invention include compounds such as N-methyl-N′-nitro-N-nitrosoguanidine and ethyl methanesulfonate. As a method for selecting a mutant strain using the mutagen, the cells of Brevibacillusformus obtained by culturing in advance are suspended in an aqueous solution of the mutagen, left for a certain period of time, and then centrifuged by a method such as centrifugation. After removing the mutagen by collecting the body, a method of culturing on a plate medium and selecting colonies of excellent strains can be exemplified. The method for selecting a colony in the selection method is not particularly limited, and a method for selecting the appearance and shape of the colony on the solid medium for spore formation (color and shape of the periphery of the colony) as an index, An example is a method of measuring the spore-forming ability by performing liquid culture after selecting and separating a large number of colonies. As a method for measuring the spore-forming ability, it is sufficient to use the property that the spore-forming ability remains resistant to stimuli such as heat when the spore-forming ability remains. After giving a stimulus such as heat to such an extent, it can be measured by examining whether it grows again after culturing.

  There is no particular limitation on the method for preserving the Brevibacillus formusus mutant of the present invention, and there are a method for preserving the activity of the fungus by subculturing in an arbitrary medium, a method for preserving by a freezing method or a freeze-drying method. It can be illustrated. In the case of the preservation method by subculturing, it is preferable to subculture to a new medium while the bacteria are actively growing. In the case of storage by freezing, it may be frozen after adding a freezing aid such as glycerol, mannitol, dimethyl sulfoxide, or the like, which is commonly used for the preservation of microorganisms, to the medium. The amount of the freezing aid added may be in a range that does not affect the survival of the bacteria. For example, in the case of glycerol, it is preferably 1/10 to 1/3 of the entire culture volume. The freezing temperature is preferably as low as possible, more preferably −20 ° C. or less, and particularly preferably −80 ° C. or less. In the case of storage by freeze-drying, it may be frozen at a low temperature as possible in advance using a freezing aid and then dried under reduced pressure. In addition to the above, skim milk or the like is also used as a freezing aid in the case of freeze-drying. The cells after lyophilization are stored at a temperature of room temperature or lower, preferably 4 ° C. or lower, more preferably −20 ° C. or lower.

  The Brevibacillus mutant strain of the present invention can be grown on a known medium suitable for culturing Bacillus bacteria. Carbon sources include molasses, glucose, fructose, maltose, sucrose, starch, lactose, glycerol, acetic acid, etc., and nitrogen sources include natural ingredients such as corn steep liquor, peptone, yeast extract, meat extract, soybean meal, and acetic acid. Amino acids such as ammonium, ammonium chloride, ammonium sulfate, aspartic acid / glycine, inorganic salts include sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphates such as dipotassium hydrogen phosphate, Sodium chloride and other metal ions include magnesium chloride, magnesium sulfate, iron (II) sulfate, iron (III) sulfate, iron (II) chloride, iron (III) chloride, iron citrate, iron iron sulfate, calcium chloride, sulfuric acid Calcium, zinc sulfate, copper sulfate, copper chloride, manganese sulfate, manganese chloride, etc. Yeast extract as an off-biotin, nicotinic acid, thiamine, riboflavin, inositol, pyridoxine, or the like can be used. In addition to carbon, nitrogen and inorganic salt sources, a suitable nutrient source may be added to the medium. If desired, it may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. When a solid medium is used, a solidifying agent such as agar or gellan gum is added to the medium having the above composition and heated to dissolve, then dispensed into a test tube or petri dish used for culture, cooled to the target temperature and solidified. It may be used after conversion. The culture temperature for Bacillus bacterium growth is 20 to 40 ° C, preferably 25 to 35 ° C, more preferably about 30 ° C. The pH of the medium is 5 to 10, preferably around 7.

  The Brevibacillus mutant strain of the present invention is characterized by a deficiency in sporulation ability. Therefore, a method for producing the protein using a recombinant Brevibacterium formusus mutant obtained by transforming an expression plasmid containing a DNA sequence encoding the protein into the mutant of the present invention is a method of producing high protein secretion of Brevibacillus formusus. It is possible to provide a production method having the ability to reduce the environmental impact of the recombinant mutant strain. In addition, when the protease activity of the recombinant mutant is reduced compared to the Brevibacterium formosus wild strain, degradation of the expressed protein derived from the protease is reduced compared to the wild strain, and thus the production amount of the protein is further increased. This is preferable. Hereinafter, the production of human Fc receptor FcγRI will be described in detail as an example of protein production using the Brevibacterium forms mutant of the present invention.

  The FcγRI-encoding polynucleotide used when the human Fc receptor FcγRI is genetically engineered using the Brevibacterium formusus mutant of the present invention includes all or part of the gene encoding the human FcγRI. However, a polynucleotide obtained by converting the codon of the polynucleotide from a human type to a Bacillus bacterium type is more preferable. As one embodiment of the codon-converted polynucleotide, a rare codon in a bacterium belonging to the genus Bacillus present in at least the 64th to 867th polynucleotide of SEQ ID NO: 1 among the genes encoding human FcγRI is encoded. And a polynucleotide in which the amino acid to be converted is converted into a codon that is frequently used in the translation mechanism of Bacillus bacteria (Japanese Patent Application No. 2008-046438). The rare codon means a codon that is used less frequently in the host. The frequency of codon usage in the host can be inferred from the analysis result of the base sequence of the genomic gene and the like. For example, as a rare codon in Brevibacterium choshinensis, a kind of bacteria belonging to the genus Bacillus, the serine (Ser) codon UCA, leucine (Leu) codon CUA, arginine (Arg) codon CGG, AGA, AGG, isoleucine (Ile) codon AUA. Information on codon usage frequency can also be obtained from a public database (http://www.kazusa.or.jp/codon/). Conversion from a rare codon to a frequently used codon is possible by converting the corresponding base sequence, and the base sequence can be converted by a known mutagenesis method such as Site-directed mutationage method. The method is a DNAWorks method (Non-patent document 5) or a Synthetic Gene Designer method (Non-patent document 6) in which a synthetic oligonucleotide and PCR are combined. In the above method, a full-length gene can be prepared by synthesizing an oligonucleotide group consisting of several tens of bases based on the amino acid sequence encoding the polypeptide and assembling the synthetic oligonucleotide by the PCR method. The codon conversion of the human FcγRI-encoding polynucleotide from the human type to the Bacillus bacterium type is performed by converting all rare codons in the human FcγRI gene sequence (SEQ ID NO: 1) from the human type to the Bacillus genus bacterial type. Alternatively, some rare codons, for example, rare codons at positions 64 to 867 in the polynucleotide sequence shown in SEQ ID NO: 1 may be converted from human to Bacillus bacteria.

  Further, the polynucleotide encoding human FcγRI encodes methionine for initiating transcription on the 5 ′ end side of the polynucleotide obtained by converting the codon of the polynucleotide encoding human FcγRI from the human type to the Bacillus genus bacterial type. May be added, or an oligonucleotide encoding a signal peptide sequence may be added to the 5 ′ end of the above-described polynucleotide. The signal peptide described here is a polypeptide for allowing a protein expressed in the cytoplasm to pass through the cell membrane and be secreted outside the cell membrane, and is usually present on the N-terminal side of the protein and after passing through the cell membrane. Cleaved by specific protease enzymes. Examples of the signal peptide include peptides of amino acid numbers 1 to 15 or 1 to 20 of SEQ ID NO: 1.

  In the production of genetically engineered human FcγRI using the Brevibacillus formusus mutant of the present invention, for the purpose of simply purifying human FcγRI, the above-described polynucleotide is encoded with a peptide serving as a tag. Oligonucleotides may be added. Examples of the tag peptide include polyhistidine tag (His-tag), Mick tag (C-myc tag) and the like. The position to be added may be on either the N-terminal side or the C-terminal side as long as the biological activity of the above-mentioned polypeptide is not impaired. Addition of an oligonucleotide encoding a tag peptide to the oligonucleotide described above can be made by genetic engineering by a method well known to those skilled in the art.

  In the production of a genetically engineered human type FcγRI using the Brevibacterium formusus mutant of the present invention, a genetically modified plasmid vector into which a human type FcγRI polynucleotide having a codon changed from a human type to a Bacillus bacterium type is inserted, A gene recombinant plasmid vector capable of expressing human FcγRI can be obtained by genetically inserting the described human FcγRI polynucleotide into an appropriate position of a known expression plasmid vector. Examples of known expression plasmid vectors include pUB110, pC194, pE194, pWVO1, etc. used for transformation of Bacillus bacteria. The appropriate position mentioned here means a position that does not destroy the replication function of the plasmid vector, a desired antibiotic marker, or a region related to transmissibility. Then, human FcγRI can be expressed by culturing a transformant obtained by transforming the above described recombinant plasmid vector into the Brevibacillus formusus mutant of the present invention.

  In the production of genetically engineered human FcγRI using the Brevibacillus formusus mutant of the present invention, the procedures and methods for introducing and expressing foreign genes into Brevibacillus formusus are not limited to the methods described in the Examples. Specific examples include those commonly used in the field of genetic engineering, and specific examples include electroporation and Tris-PEG.

  In the production of genetically engineered human FcγRI using the Brevibacillus formusus mutant of the present invention, the composition of the medium used for the production of human FcγRI is that the mutant of the present invention grows and produces human FcγRI. It can be used as long as it can be used, and may be appropriately selected from the carbon sources, inorganic salts, and vitamins described above. In a preferred embodiment, a selective agent based on the construction of the expression plasmid vector may be included in the medium in order to selectively allow the growth of Brevibacillusformus containing the expression plasmid vector. For example, neomycin is added to the medium for the growth of cells expressing the neomycin resistance gene. When using a promoter derived from the cell wall protein of the mutant strain of the present invention as an expression plasmid vector, since the growth of the bacteria works actively after entering the stationary phase, the turbidity of the culture solution (absorbance at 600 nm) is measured, After the transition from the logarithmic growth phase to the stationary phase, the protein can be secreted and expressed in the culture solution by subsequent culturing. The culture temperature is preferably 15 to 40 ° C., and the pH is preferably 6 to 8. In addition, the culture time may be any time as long as FcγRI is sufficiently produced, and is usually set between several hours and 200 hours, but the optimum culture time depends on conditions such as medium components, culture temperature, and aeration rate. In order to change, it is preferable to determine by measuring the expression level and activity of the expressed protein.

  In order to obtain human-type FcγRI from the culture solution, an extraction method may be appropriately selected depending on the form of expression. When expressed in the culture supernatant, the cells are separated by centrifugation, and human-type FcγRI may be extracted from the resulting culture supernatant. When expressed in the cytoplasm, the bacterial cells are collected by centrifugation, and the bacterial cells are disrupted by adding an enzyme treatment agent, a surfactant or the like, and human FcγRI can be extracted. In order to separate and purify human FcγRI from the extracted protein, liquid chromatography can be used. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like. A high-purity human FcγRI can be prepared by performing a purification operation by combining these chromatographies.

  The method for analyzing human FcγRI obtained by genetic engineering techniques using the Brevibacterium formusus mutant of the present invention is not particularly limited as long as it can be stably and efficiently quantified from the culture solution. However, ELISA method (enzyme-linked immunosorbent method) ) Is preferable in terms of simplicity.

  Human FcγRI obtained by genetic engineering techniques using the Bacillus bacterium mutant of the present invention is used in various applications such as pharmaceuticals, clinical test drugs, biosensors, or affinity ligands (separating agents). The form and purity at the time of use vary depending on the application, and it can be used as it is in the culture solution, can be used after highly purified, and is used at various purification levels having intermediate purity. The purified human FcγRI is used as a pharmaceutical, a clinical test agent, a biosensor, an affinity ligand (separating agent) or the like. The purity required for purified human type FcγRI varies depending on its use, and it is prepared and used to the purity required for each purpose.

  The mutant strain of the present invention can be obtained by treating a Brevibacterium formosus wild strain with a mutagen. Since the mutant strain of the present invention is deficient in sporulation ability, it does not have the ability to survive in the natural environment. Therefore, when the mutant strain of the present invention is used as a host for producing a protein by genetic engineering, the mutant strain has a high protein secretion ability that is characteristic of Brevibacillus formalus, and the mutant strain has little environmental impact. A method can be provided.

  In addition, when the protease activity of the mutant strain is lower than that of the wild type strain of Brevibacillus formosus, the degradation of the expressed protein derived from the protease is reduced compared to the wild type strain. It can be produced efficiently.

  In particular, the mutant strain of the present invention is preferable as a host for genetic engineering production of the human type Fc receptor FcγRI, and the obtained FcγRI can be used as a ligand for pharmaceuticals, clinical diagnostic agents, biosensors, or separating agents.

It is a figure which shows the structure of human type | mold Fc receptor FcγRI.

  Hereinafter, embodiments of the present invention will be described in detail. Needless to say, the present invention is not limited to these examples, and can be arbitrarily changed without departing from the scope of the invention.

Example 1 Income of Mutant of the Present Invention (Part 1)
Brevibacillus formusus mutants lacking the ability to form spores were obtained by the method described below.

  (1) Brevibacillus formalus (NBRC15716) is inoculated into a 14 mL tube containing 4 mL of LB medium (tryptone 10 g / L, salt 10 g / L, yeast extract 5 g / L), and cultured overnight at 37 ° C. and 150 rpm. I did it. 1.0 mL of this culture solution was transferred to a 1.5 mL Eppendorf tube, and the cells were collected by centrifugation at 15000 rpm for 10 minutes.

  (2) A 0.1 M phosphate buffer solution (pH 7.2) of 10 μg / mL N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter abbreviated as NTG) Suspended in 0 mL and allowed to stand at room temperature for 10 to 60 minutes for mutagenesis treatment.

  (3) Centrifugation collect | recovered the microbial cells, The operation which resuspends in a 0.1M phosphate buffer (pH 7.2) was repeated twice, and NTG was removed.

  (4) 2 mL of sporulated medium (nutrient broth 8 g / L, potassium chloride 1 g / L, magnesium sulfate heptahydrate 120 mg / L, calcium nitrate tetrahydrate 236 mg / L, chloride Manganese tetrahydrate 198 mg / L, iron sulfate heptahydrate 0.278 mg / L, pH 7.0) was suspended and cultured overnight at 37 ° C. with shaking.

  (5) Appropriately dilute the culture solution of (4) and apply an appropriate amount to a spore-forming solid medium (medium solidified after adding 14 g of agar to 1 L of spore-forming medium and dissolving) And static culture at 37 ° C. for 3 days.

  (6) A transparent colony or a wet or creamy colony was selected from the resulting colonies, and after staining, a mutant strain lacking the ability to form spores was obtained by observing under a microscope. .

(7) It was confirmed by the staining method (Wirtz method) using malachite green and safranin that the mutant strain obtained in (6) did not produce spores.
(7-1) The colony of the mutant strain selected in (6) was suspended in about 50 μL of water dropped on a slide glass, dried, and lightly exposed to flame to immobilize.
(7-2) 200 μL of a 5% malachite green aqueous solution was dropped on the immobilized colonies, and heated and stained for 3 minutes.
(7-3) After cooling, the excess colony green aqueous solution was washed away by washing so that the immobilized colonies were not washed away, and 200 μL of 0.5% safranin solution was added dropwise, followed by staining for 30 seconds.
(7-4) After washing with water and drying, the degree of staining of bacterial cells and spores was observed with a microscope, and it was confirmed that there were no spores stained in green.

(8) It was confirmed by the following heat treatment method that the mutant strain obtained in (6) lacked the spore-forming ability.
(8-1) After culturing the mutant strain selected in (6) in a spore-forming solid medium, the resulting colonies were suspended in 500 μL of physiological saline and heat-treated at 80 ° C. for 20 minutes. If the spore-forming ability remains, spore is formed to survive the heat treatment, and if the spore-forming ability is deficient, it is killed by the heat treatment.
(8-2) Bacillus subtilis 168 strain showing spore-forming ability as a negative control strain, Brevibacillus choshinensis SP3 strain (manufactured by Takara Bio Inc.) commercially available as a spore-forming ability-deficient strain as a positive control strain (8- It processed by the method similar to 1).
(8-3) After coating on the LB solid medium, it was confirmed whether or not the spore-forming ability was lost by stationary culture at 37 ° C.

  Brevibacillus formusus mutant strain (L-D7-HK1 strain) obtained in (6), Bacillus subtilis strain 168 (with spore-forming ability), Brevibacterium chosinensis SP3 strain (without spore-forming ability), and Brevibacillus 15 wild-type strain 16 Table 1 shows the results of summarizing the growth situation after heat treatment (with spore formation ability). Since the L-D7-HK1 strain did not grow by heat treatment at 80 ° C. for 20 minutes, it was confirmed that the L-D7-HK1 strain was deficient in sporulation ability.

実施例２ Ｌ−Ｄ７−ＨＫ１株の保存
（１）実施例１で得られたＬ−Ｄ７−ＨＫ１株をＬＢ培地で培養した。得られた培養液１ｍＬに、滅菌した７０％グリセロ−ル水溶液０．４ｍＬを加えてよく混合後、あらかじめ滅菌したサンプル瓶（容量２ｍＬ）に分注することでグリセロールストックを調製した。前記グリセロールストックは−８０℃にて凍結保存した。
（２）ＬＢ培地プレート上にＬ−Ｄ７−ＨＫ１株を植菌して３７℃で静置培養後、生じたコロニーを２０％スキムミルク水溶液で懸濁した。次いで、アンプル管に約０．１ｍＬずつ分注して、液体窒素中で凍結した後に凍結乾燥機で乾燥し、減圧を保ったまま封管することで凍結乾燥菌体を調製した。

Example 2 Storage of L-D7-HK1 strain (1) The L-D7-HK1 strain obtained in Example 1 was cultured in an LB medium. A glycerol stock was prepared by adding 0.4 mL of a sterilized 70% aqueous glycerol solution to 1 mL of the obtained culture and mixing well, and then dispensing into a pre-sterilized sample bottle (volume 2 mL). The glycerol stock was stored frozen at -80 ° C.
(2) The L-D7-HK1 strain was inoculated on the LB medium plate, and after standing at 37 ° C., the resulting colony was suspended in a 20% skim milk aqueous solution. Next, about 0.1 mL each was dispensed into an ampoule tube, frozen in liquid nitrogen, dried with a freeze dryer, and sealed with the vacuum maintained to prepare freeze-dried cells.

Example 3 FcγRI production using the mutant strain of the present invention The protease activity possessed by the mutant strain (L-D7-HK1 strain) obtained in Example 1 and Brevibacterium formosus wild strain (NBRC15716), and the two strains The expression level of human Fc receptor FcγRI when used as a host was compared.

  (1) The FcγRI gene (with a C-myc tag for purification and labeling added to the C-terminal side of the gene) is wild-type or carried out by a general genetic engineering technique using a plasmid vector. After transformation into the mutant strain obtained in Example 1 (L-D7-HK1 strain), BTYm2 medium (polypeptone 30 g / L, yeast extract 7.5 g / L, glucose 15 g / L, ferric sulfate heptahydrate 0.01 g / L, manganese sulfate tetrahydrate 0.01 g / L, zinc sulfate heptahydrate 0.001 g / L) was inoculated into 200 mL, and cultured at 30 ° C. for 2 days.

(2) After collecting the centrifugal supernatant, the protease activity in the supernatant was measured by the casein method shown below.
(2-1) 0.2 mL of a 0.2 M phosphate buffer solution (pH 7.2) containing 1% (w / v) casein was heated at 30 ° C. for 5 minutes, and then an enzyme solution in which the enzyme concentration was appropriately adjusted ( Culture supernatant) 0.2 mL was added, and the reaction was performed at 30 ° C. for about 10 minutes to half a day.
(2-2) The reaction was stopped by adding 0.4 mL of a reaction stop solution (0.1 M trichloroacetic acid, 0.2 M sodium acetate, 0.3 M acetic acid), left at 30 ° C. for 30 minutes, and then acid-denatured. The protein was centrifuged (15000 rpm, 10 minutes).
(2-3) Dispensing 0.2 mL of the centrifuged supernatant, 1.0 mL of an alkaline solution (0.4 M sodium carbonate) and ferrin reagent (phenol reagent diluted 5 times with deionized water) 0.2 mL And heated at 30 ° C. for 20 minutes.
(2-4) Absorbance at 660 nm was measured using a spectrophotometer to determine the amount of acid-soluble protein degradation product produced. The absorbance per turbidity of the bacteria was calculated and the protease activity was compared. In addition, after adding a reaction stop solution to said enzyme reaction system, let the system which added the enzyme solution be a blank.

(3) After recovering the centrifugal supernatant, the FcγRI degradation activity in the supernatant was measured by the method described below.
(3-1) A mixture of 0.675 mL of the centrifugal supernatant of the culture solution and 0.075 mL of a suitably diluted purified FcγRI solution, and then allowed to stand at 4 ° C. and 30 ° C. Was done.
(3-2) The residual ratio of FcγRI with respect to changes over time was measured by the ELISA method shown below, and the FcγRI degradation activity in the culture supernatant was measured.
(3-2-1) A gamma globulin preparation (manufactured by Chemo-Serum Therapy Laboratories) diluted to 50 μg / mL was added to each well in a 96-well plate (manufactured by Nunc), and 100 μL was added to each well and allowed to stand overnight at 4 ° C. It was fixed by doing.
(3-2-2) After washing with TBS buffer solution (Tris-HCl buffer solution (pH 8.0) containing 0.2% (w / v) Tween 20, 150 mM NaCl), Starting Block Blocking Buffers (manufactured by PIERCE) ) To perform a blocking operation.
(3-2-3) After washing with TBS buffer, 100 μL of the culture supernatant of the transformant was added and reacted with the immobilized antibody gamma globulin (30 ° C., 2 hours). After completion of the reaction, the reaction mixture was washed with TBS buffer and Anti-c-myc-HRP antibody (Meltenyi Biotec BmgH) was added.
(3-2-4) After completion of the reaction, the mixture was washed with TBS buffer, TMB Peroxidase Substrate (manufactured by KPL) was added, and the absorbance at 450 nm was measured.

  (4) After collecting the centrifugal supernatant, the FcγRI production amount in the supernatant was measured by the ELISA method shown in (3-2), and the FcγRI productivity in the transformant was measured.

  The results are shown in Table 2. The numbers in parentheses indicate the relative value (%) of the L-D7-HK1 strain when the result of the wild strain is 100%. The protease activity of the L-D7-HK1 strain was 7.61 (protease activity per turbidity, hereinafter the same), which was about 70% lower than the activity of the wild strain (24.8). Further, when compared with the FcγRI degradation activity, the FcγRI remaining rate after 24 hours at 30 ° C. was 36% in the wild strain, and 76% in the L-D7-HK1 strain, showing an inhibitory effect on protease activity. As described above, the L-D7-HK1 strain obtained in Example 1 was found to be a strain that lacks the spore-forming ability and has a protease activity lower than that of the wild strain.

  When compared with the expression level of FcγRI, it was 77 μg / L when the wild strain was used as the host, whereas the expression level of FcγRI when using the L-D7-HK1 strain as the host was 80 μg / L. It was better than the stock. As described above, since the L-D7-HK1 strain has a protease activity lower than that of the wild strain, degradation of the expressed protein derived from the protease is reduced. Therefore, it is considered that the expression level of FcγRI was improved.

Claims (5)

A Brevibacillus formusus mutant strain deficient in sporulation ability, obtained by treating a Brevibacillus formosa wild strain (NBRC15716) with a mutagen. Furthermore, the mutant strain of Claim 1 in which protease activity fell compared with Brevibacterium formosus wild strain (NBRC15716). A recombinant Brevibacillus formusus strain capable of expressing the protein, obtained by transforming an expression plasmid containing a DNA sequence encoding the protein into the mutant strain according to claim 1 or 2. The recombinant according to claim 3, wherein the protein is human Fc receptor FcγRI. A method for producing a human Fc receptor FcγRI using the recombinant according to claim 4.

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