JP2015077152A - Method for producing recombinant protein by using genetically engineered brevibacillus bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel condition for culturing Brevibacillus bacterium, which enables the improvement in the quantity of a recombinant protein secreted/produced per cell and the increase in the density of cells during the culture and also enables the preparation of the recombinant protein at a lower cost compared with conventional techniques, and which can be scaled up to an industrial level.SOLUTION: In the production of a recombinant protein by using a genetically engineered Brevibacillus bacterium, the culture of the bacterium is carried out by using fructose as a carbon source, thereby providing a novel condition for culturing the Brevibacillus bacterium, which enables the improvement in the quantity of the recombinant protein secreted/produced per cell and the increase in the density of cells during the culture and also enables the preparation of the recombinant protein at a lower cost compared with conventional techniques, and which can be scaled up to an industrial level.

Description

The present invention relates to a method for producing a recombinant protein using a recombinant Brevibacillus bacterium.

Production of recombinant proteins using Brevibacillus bacteria as a host is used for the production of various heterologous proteins (Non-patent Document 1). Various culture conditions have been studied to improve the secretory production of recombinant protein per cell and to increase the density of cells during culture. As a result, glucose has been used as the main carbon source of the medium. Sucrose, glycerin and the like are usually used. However, it has not been known so far that fructose is used as a suitable carbon source for improving the secretory production amount and increasing the density of the cells, although it is the same monosaccharide as glucose.

This is thought to be caused by the fact that fructose is more rapidly colored in the presence of amino acids than glucose (Non-patent Document 2), so that the culture supernatant is easily colored. In particular, in the production of recombinant proteins by the Brevibacillus bacterium, which is a secretory expression system, the colored substance derived from the fructose binds to the expressed recombinant protein, and the protein to which the colored substance is bound is treated as an impurity derived from the target substance in the purification process. Need to be removed. The generation of these impurities is presumed to prevent the use of fructose as the main carbon source because it results in a significant reduction in the yield of the target product and is difficult to remove due to its approximate properties.

Thus, although production of recombinant proteins using Brevibacillus bacteria as a host has been conducted under various culture conditions, under the conventional conditions, depending on the recombinant protein to be expressed, high secretory expression was reached. In other cases, superiority to other host vector systems could not be found.

By the way, among protein drugs produced using genetic recombination techniques, the demand for antibody drugs is rapidly expanding. Antibody drugs are mainly produced using cultured CHO cells as glycoproteins of about 150 kDa.

In the manufacture of antibody pharmaceuticals, affinity chromatography having antibody binding ability is generally used, and the most commonly used protein such as protein A, protein G and protein L is immobilized on an appropriate resin. Chromatography with a carrier. Among the proteins used as ligands for these purification carriers, protein A is particularly frequently used.

On the other hand, an affinity carrier having a protein as a ligand is required to have high quality as a material for producing a pharmaceutical product. Protein ligands themselves are also required to have the same level of quality as protein pharmaceuticals, and it is not possible to inexpensively supply affinity carriers using these as raw materials. These affinity carriers account for a large proportion of the antibody drug production cost, which is a major impediment to reducing the cost of antibody drug products. Therefore, a method for procuring these ligand proteins with high quality at low cost has been desired.

In order to establish a technique for stably producing a large amount of a partial sequence of protein A so far, the present inventors have used a Brevibacillus bacterium as a host to efficiently produce a large amount of the partial sequence of protein A into a culture solution. Has been found to be secreted and stably accumulated, and can be easily separated and recovered with high purity (Patent Document 1).

In addition, among recombinant proteins produced using genetic recombination techniques, the demand for physiologically active proteins such as insulin is also expanding. One such bioactive protein is feline proinsulin. Purified feline proinsulin has not been put on the market so far, and the reliability of proinsulin measurement at the time of diagnosis of feline diabetes has been low, which has prevented early detection of feline diabetes. In addition, other types of insulin are used as therapeutic agents for diabetes, and it has been desired to develop an industrial production method of cat proinsulin as a raw material for cat insulin. Therefore, in recent years, as a method for producing feline proinsulin, a technique for expressing feline proinsulin in Escherichia coli has been constructed, and inclusion bodies are formed in Escherichia coli during the culture (Non-patent Document 3). Forming inclusion bodies during culture increases the load on the purification process, resulting in higher product costs. Therefore, realization of an economical production method of feline proinsulin in a secretory expression system is strongly desired.

WO2006 / 004067

Protein Nucleic Acid Enzyme Feb; 37 (3 Suppl): 258-68, (1992) `` Food discoloration and its chemistry '' p248-249, (1967), Koyo Shoin (Tokyo) Domestic Animal Endocrinology, 30: 28-37, (2006)

In the present invention, when producing a recombinant protein using a recombinant Brevibacillus bacterium, the secretory production of the recombinant protein per cell is improved and the density of the cell at the time of cultivation is increased compared to the conventional techniques. It is an object of the present invention to provide new culture conditions for Brevibacillus bacteria that can be achieved and can be prepared at a lower cost and can be scaled up to an industrial scale.

As a result of intensive studies to solve the above-mentioned problems, the present inventors surprisingly cultivate using, as a carbon source, fructose that has been avoided so far due to the ease of browning. Thus, the secretory production amount of the recombinant protein per microbial cell was improved, and the microbial cell during the cultivation was found to have a higher density, thereby completing the present invention.

That is, the present invention provides a method for producing a recombinant protein, which comprises culturing using fructose as a carbon source when producing a recombinant protein using a recombinant Brevibacillus bacterium. It is.

According to the present invention, in the production of recombinant white matter using recombinant Brevibacillus bacteria, the secretory production amount of the recombinant protein per cell is improved, and the cell density during culture is increased, It is possible to achieve the productivity of recombinant protein more than 3 times the conventional.

It is a figure which shows the partial arrangement | sequence (SPA ') expression vector (Spa'-pNK3260) of protein A based on Example 2 of this invention. The DNA sequence which codes the promoter sequence of the partial arrangement | sequence expression vector (Spa'-pNK3260) of protein A (SPA ') based on Example 2 of this invention, Shine-Dalgarno sequence, a signal peptide, and protein A (SPA') is shown. FIG.

The present invention achieves high secretion expression and / or high density of cells during culture by using fructose as a carbon source when producing recombinant proteins using recombinant Brevibacillus bacteria. Is. The present invention will be described in detail below.

A recombinant Brevibacillus bacterium refers to a bacterium obtained by inserting a DNA encoding a recombinant protein into an expression vector and transforming the expression vector into a host Brevibacillus bacterium.

The DNA encoding the recombinant protein may be any DNA as long as the amino acid sequence obtained by translating the base sequence of the DNA constitutes the target recombinant protein. Such a DNA sequence can be obtained by using a commonly known method such as a polymerase chain reaction (hereinafter abbreviated as PCR) method. It can also be synthesized by a known chemical synthesis method (Nucleic acids Res., 1984, 12: 4359), and can also be obtained from a DNA library.

The DNA sequence may have a codon substituted with a degenerate codon and need not be identical to the original DNA sequence as long as it encodes the same amino acid when translated in the genus Brevibacillus .

The expression vector includes a DNA sequence encoding the recombinant protein or a partial sequence thereof, and a promoter capable of functioning in Brevibacillus bacteria operably linked to the sequence. The promoter is not limited as long as it can function in bacteria of the genus Brevibacillus, but Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus A promoter derived from a bacterium such as (Corynebacterium) and operable in a bacterium belonging to the genus Brevibacillus is preferred. Brevibacillus bacteria cell wall protein, middle wall protein (MWP) and outer wall protein (OWP) (Udaka, S. et al. Method Enzymol., 1993, 217: 23-33) or Brevibacillus choshinensis HPD31 More preferred is a promoter of a gene encoding the cell wall protein HWP (J. Bacteriol., 1990, 172: 1312-1320).

In addition, the expression vector preferably further includes a Shine-Dalgarno sequence (SD sequence) and a signal sequence that can function in Brevibacillus bacteria downstream of the promoter. The expression vector may optionally include a marker sequence.

The SD sequence following the above promoter is derived from bacteria such as Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus SD sequence operable in Brevibacillus genus bacteria The SD sequence present in the upstream of the gene encoding MWP, OWP or HWP is more preferable.

The DNA encoding the secretory signal peptide following the SD sequence is not particularly limited as long as it encodes the following secretory signal peptide, and encodes the same amino acid when translated in Brevibacillus brevis. As long as it is, it does not have to be the same as the original base sequence. Examples of the secretory signal peptide are derived from bacteria such as Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus, Corynebacterium genus, and the like in the genus Brevibacillus genus The secretory signal peptide that can be operated is preferable, and the secretory signal peptide of MWP, OWP, or HWP is more preferable.

Moreover, what improved the amino acid sequence of the conventional secretion signal peptide may be used. For example, the signal peptide of MWP, Met-Lys-Lys-Val-Val-Asn-Ser-Val-Leu-Ala-Ser-Ala-Leu-Ala-Leu-Thr-Val-Ala-Pro-Met-Ala-Phe -Ala (SEQ ID NO: 1) is converted to Met-Lys-Lys- Arg-Arg -Val-Val-Asn-Ser-Val- Leu-Leu-Leu-Leu-Leu-Leu- Ala-Ser-Ala-Leu-Ala -Leu-Thr-Val-Ala-Pro-Met-Ala-Phe-Ala (SEQ ID NO: 2) may be a secretory signal peptide to which basic or hydrophobic amino acid residues are added, as indicated by the underlined portion. Moreover, the secretion signal peptide conventionally used in the secretory protein of the genus Brevibacillus may be used. Further, a signal peptide inherently possessed by the recombinant protein to be expressed may be used.

DNA encoding the above promoter, SD sequence, and secretory signal peptide can be obtained from, for example, Brevibacillus bacteria. Preferably, Brevibacillus brevis 47 strain (FERM BP-1223), Brevibacillus brevis 47K strain (FERM BP-2308), Brevibacillus brevis 47-5 strain (FERM BP-1664), Brevibacillus choshinensis HPD31 A known PCR using a chromosomal DNA of a strain (FERM BP-1087), a Brevibacillus choshinensis HPD31-S strain (FERM BP-6623), or a Brevibacillus choshinensis HPD31-OK strain (FERM BP-4573) as a template It can be obtained by specifically increasing by the method.

In the expression vector, any of the above promoters, any of the above SD sequences, any DNA encoding any of the above signal peptides, and DNA encoding the recombinant protein can operate in Brevibacillus bacteria. It is preferable that it is connected to.

As the vector, a plasmid vector is preferable. Specific examples of plasmid vectors useful for the expression of genes of the genus Brevibacillus include, for example, pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP-A-2-31682), pNY700 (JP-A-4-278091). Publication), pHY4831 (J. Bacteriol. 1987. 1239-1245), pNU200 (Shigezo Takataka, Journal of Japanese Society of Agricultural Chemistry 1987. 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30:75). -80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU211R2L5 (JP-A-7-170984), pNH301 (Appl. Environ. Microbiol., 1992. 58: 525-531), pNH326, pNH400 (J. Bacteriol., 1995. 177: 745-749), pHT210 (JP-A-6-133382), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363), or Escherichia coli Breviva PNCO2 (Japanese Patent Laid-Open No. 2002-23869), which is a shuttle vector with a bacterium belonging to the genus Chilus, can be used, but is not limited thereto.

The above plasmid vector can be prepared by those skilled in the art based on literature information. In addition, an expression vector containing a promoter that functions in a bacterium of the genus Brevibacillus, an SD sequence, and a DNA encoding the target protein, or a gene fragment containing each of these nucleotide sequences is directly incorporated into a chromosome and expressed (specifically (Kaihei 9-135893) may be used. In such a method, a known method already used in Bacillus subtilis or yeast can be transferred to Brevibacillus bacteria.

When a recombinant protein is produced in a secreted form, it is preferable to add or link DNA encoding a signal peptide that functions in the genus Brevibacillus upstream of the DNA encoding the polypeptide.

Arbitrary Brevibacillus bacteria can be used as a host cell used for obtaining a transformant. Brevibacillus bacteria include, but are not limited to, Brevibacillus agri, Brevibacillus bolsterensis, Brevibacillus brevis, Brevibacillus centroporus, Brevibacillus choshinensis, Brevibacillus formosas, Brevibacillus invocatus, Brevibacillus・ Includes Latinosporus, Brevibacillus limnophilus, Brevibacillus parabrevis, Brevibacillus reuszeli, Brevibacillus thermolver, etc.

Preferably, the bacterium belonging to the genus Brevibacillus is Brevibacillus brevis 47 strain (FERM BP-1223), Brevibacillus brevis 47K strain (FERM BP-2308), Brevibacillus brevis 47-5 strain (FERM BP-1664), Brevibacillus brevis 47-5Q strain (JCM8975), Brevibacillus choshinensis HPD31 strain (FERM BP-1087), Brevibacillus choshinensis HPD31-S strain (FERM BP-6623), Brevibacillus choshinensis HPD31-OK Strain (FERM BP-4573) and Brevibacillus choshinensis SP3 strain (Takara). In particular, the aforementioned Brevibacillus brevis 47 strain, Brevibacillus brevis 47-5Q strain, Brevibacillus choshinensis HPD31 strain, and Brevibacillus choshinensis HPD31-S strain are suitable.

Brevibacillus brevis 47-5Q strain (JCM8975) can be obtained from RIKEN BioResource Center Microbial Materials Development Office (JCM) (2-1 Hirosawa, Wako, Saitama 351-0198).

A mutant strain such as a protease-deficient strain or a high-expressing strain of the aforementioned Brevibacillus bacterium may be used depending on the purpose such as improvement of the production amount. Specifically, Brevibacillus choshinensis HPD31-derived protease mutant Brevibacillus choshinensis HPD31-OK (FERM BP-4573), and similarly Brevibacillus choshinensis HPD31-derived spore-forming ability, Brevibacillus choshinensis SP3 strain (manufactured by Takara), which is a protease mutant, and Brevibacillus brevis 47K (FERM BP-2308) obtained as a human salivary amylase high-producing strain can be used. Moreover, you may use the variant of either strain | stump | stock contained in the said Brevibacillus genus bacteria group.

Transformation of host cells of Brevibacillus genus bacteria used in the present invention can be performed by the known Takahashi et al. Method (J. Bacteriol., 1983, 156: 1130-1134) or the Takagi et al. Method (Agric. Biol. Chem. , 1989, 53: 3099-3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61: 202-203).

When a heterologous protein is highly expressed in microorganisms including Brevibacillus bacteria, it often forms an inactive protein without being correctly folded, especially when a protein with many disulfide bonds is highly expressed. Insoluble in many cases. On the other hand, when expressing the target protein, it is known that the insolubilization of the target protein and the decrease in the secretion efficiency can be suppressed by acting chaperone protein, disulfide bond isomerase and / or proline isomerase, etc. . A widely attempted method is a method in which a protein having disulfide redox activity such as PDI (protein disulfide isomerase) and / or DsbA is allowed to act (JP-A 63-294996, JP-A-5-336986). is there.

Furthermore, a method for producing a protein having a correct disulfide bond by introducing a gene encoding a protein having disulfide redox activity into a host organism and simultaneously expressing the target protein and a protein having disulfide redox activity is also known. (JP 2000-83670 A, JP 2001-514490 A, etc.).

In the case of expression of a recombinant protein according to the present invention or a protein comprising a partial sequence thereof, the expression of the protein is performed in order to reduce the burden on the host cell due to excessive protein synthesis and to facilitate protein secretion. In this case, it is possible to co-express several types of chaperone proteins, disulfide bond oxidoreductases, and / or enzymes that promote folding, such as disulfide isomerase.

For example, DsbA of E. coli (Cell, 1991, 67: 582-589, EMBO. J., which is involved in protein disulfide bonds and is considered to be an analog of protein disulfide isomerase when the protein is expressed in Brevibacillus bacteria. 1992, 11: 57-62.) And / or chaperone proteins such as DnaK, DnaJ, and GrpE (Japanese Patent Laid-Open No. 9-180558) can be expressed simultaneously. In addition, an enzyme PDI (Japanese Patent Application No. 2001-567367) and a disulfide oxidoreductase (Japanese Patent Application Laid-Open No. 2003-169675) (Molecular Microbiology, 1993, 8: 727-737) that are involved in accurate disulfide bonds of polypeptides. ) And / or an enzyme that promotes folding, such as disulfide isomerase, can be expressed at the same time as the protein to further improve secretion efficiency.

The medium used for the cultivation of the recombinant Brevibacillus bacterium is not particularly limited as long as it contains fructose as long as it can produce the recombinant protein with high efficiency and high yield. Specifically, a known carbon source or nitrogen source such as glucose, sucrose, glycerol, polypeptone, meat extract, yeast extract, casamino acid, and amino acid can be used. In addition, inorganic salts such as potassium salt, sodium salt, phosphate, magnesium salt, manganese salt, zinc salt and iron salt may be added as necessary.

In addition, if necessary, anti-foaming effects such as soybean oil, lard oil, surfactants, etc., or changing the permeability of the cell membrane material, is expected to improve the production of recombinant protein per cell A compound to be prepared may be added. The use of a surfactant is preferable because the effect of the present invention may be enhanced. The surfactant is not particularly limited as long as it does not adversely affect the growth of recombinant Brevibacillus bacteria and / or recombinant protein production, and is preferably a polyoxyalkylene glycol surfactant.

When an auxotrophic host cell is used, a nutrient substance required for growth may be added. If necessary, antibiotics such as penicillin, erythromycin, chloramphenicol and neomycin may be added.

Furthermore, various known protease inhibitors may be added at an appropriate concentration in order to suppress degradation of the target protein by the host-derived protease present inside and outside the cell body and to lower the molecular weight. Examples of protease inhibitors include phenylmethane sulfonyl fluoride (PMSF), Benzamidine, 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF), Antipain, Chymostatin, Leupeptin, Pepstatin A, Phosphoramidon, Aprotinin, Ethylenediaminetetra acetic acid (EDTA) Other examples include commercially available protease inhibitors.

As a method for adding fructose, which is added in order to improve and achieve secretion production of recombinant protein per cell and / or achieve high density of cells during culture, the initial concentration is 1%. Above or 9% or less is preferable, more preferably 1 to 9% of the initial. More preferably, fructose is added in a timely manner so that the fructose concentration is 9% or less, particularly 4% or less during the culture. Fructose addition methods include divided or continuous addition. Examples of such a method include, but are not limited to, a method of adding fructose after 6 hours from the start of culture.

The antibody-binding protein is not a protein that is recognized as an antigen by an antibody, but is a protein that can bind to a portion other than the antigen recognition site (for example, an Fc portion) of an antibody. The structure is not particularly limited as long as it is a protein that can bind to a site different from the antigen recognition site of the antibody. Examples of such proteins include protein A, protein G, and protein L.

Protein A is one of cell wall proteins produced by the Gram-positive bacterium Staphylococcus aureus, and has a molecular weight of about 42,000. Its structure consists of seven functional domains (signal sequence S from the amino terminus, immunoglobulin binding domain E, immunoglobulin binding domain D, immunoglobulin binding domain A, immunoglobulin binding domain B, immunoglobulin binding domain C, Staphylococcus aureus) Bacterial cell wall binding domain X) (Proc. Natl. Acad. Sci. USA, 1983, 80: 697-701, Gene, 1987, 58: 283-295, J. Bio. Chem., 1984, 259 : 1695-1702).

The relative affinity of this protein A for the immunoglobulin binding domain is as follows: pH, Staphylococcus aureus strain (Infec. Immun., 1987, 55: 843-847), and immunoglobulin classes (IgG, IgM, IgA, IgD, IgE) and subclasses (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) are known to depend on many factors, particularly in the immunoglobulin class, human IgG1, human IgG2, human IgG4 and mouse IgG2a, It shows strong binding to the Fc part of mouse IgG2b and mouse IgG3.

Protein G is a kind of cell wall protein produced by Streptococcus of Group C and G, and has a molecular weight of about 59,000. The structure consists of five functional domains (from the amino terminus, signal sequence SS, albumin binding domain by repetition of sequences A and B, immunoglobulin binding domain by repetition of sequences C and D, cell wall transmembrane domain W, and transmembrane domain. M).

The immunoglobulin-binding domain of protein G exhibits extensive binding to the Fc portion of mammalian IgG compared to that of protein A (J. Immunol., 1984, 133: 969-974, J. Biol. Chem. , 1991, 266: 399-405).

Protein L is one of the proteins produced by Peptostreptococcus magnus and has a molecular weight of about 79,000. The structure consists of 6 functional domains (from the amino terminus, signal sequence SS, amino terminal domain A, immunoglobulin binding domain B, 5 repeats, unknown function domain 2 repeats, transmembrane domain W, transmembrane domain M) It is composed of

The immunoglobulin binding domain of this protein L binds to the kappa light chain of immunoglobulin. (J. Biol. Chem., 1989, 264: 19740-19746, J. Biol. Chem., 1992, 267: 12820-12825).

“Partial sequence” of protein A, protein G, and protein L is a protein that is composed of an arbitrary part of amino acid sequences constituting protein A, protein G, and protein L and has antibody binding activity. Specifically, for example, the amino acid sequence shown after the 31st Ala of SEQ ID NO: 9 obtained by removing the above-mentioned signal sequence S and cell wall binding domain X from protein A (see “SPA of Example 1”). Corresponds to '". See FIGS. 1 and 2, SEQ ID NOS: 8 and 9.) corresponds to the“ partial sequence ”of protein A. A peptide having an antibody binding activity obtained by removing one or a plurality of immunoglobulin binding domains is also included in the “partial sequence” as long as it has an antibody binding activity.

“Functional variants and their linking sequences” of protein A, protein G and protein L are amino acids in the state in which at least one of the immunoglobulin binding domains of protein A, protein G and protein L retains antibody binding activity. This refers to antibody-binding proteins that have been subjected to substitution, insertion, and deletion treatments, and have changed sensitivities to physicochemical environmental factors such as drugs, enzymes, heat, and pH, and constructs that are homozygous or heterozygically linked. The combination and the number of connections are not limited. For example, a sequence in which the 29th Gly of the C domain of protein A is modified to Ala and linked five times (corresponding to “C-G29A” in Example 6; see SEQ ID NO: 10) is the above-mentioned “functional mutant and These linkage sequences are exemplified.

The physiologically active protein is a protein used as a pharmaceutical active ingredient, and specifically includes peptide hormones, cytokines, growth factors, hematopoietic factors, enzymes, and precursors thereof.

Peptide hormones are peptides that are produced and secreted by endocrine cells in animal tissues in accordance with information inside and outside the body, are transported to the target cells by the bloodstream, and regulate the activity of the target cells as foreign signals. Examples of peptide hormones or precursors thereof include insulin, proinsulin, or preproinsulin, particularly feline insulin, feline proinsulin, or feline preproinsulin.

Insulin is a peptide hormone that is synthesized as proinsulin which is a biosynthetic precursor in the rough endoplasmic reticulum of pancreatic B cells, is converted into insulin, is stored in B granules, and is released into the blood in response to secretory stimulation. is there.

Cat proinsulin is the biosynthetic precursor of cat insulin.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these. In this implementation, production and manipulation of the recombinant DNA were performed according to the following experiment document unless otherwise specified. (1) T. Maniatis, EF Fritsch, J. Sambrook, “Molecular Cloning / A Laboratory Manual”, 2nd edition (1989), published by Cold Spring Harbor Laboratory (USA) . (2) Edited by Masaaki Muramatsu “Lab Manual Genetic Engineering”, 3rd edition (1996), published by Maruzen Co., Ltd. In addition, commercially available products were used for reagents, restriction enzymes and the like used in this example unless otherwise specified.

Example 1 Construction of Brevibacillus Expression Vector pNK3260 The MWP P5 promoter contained in pNH326 (J. Bacteriol., 1995, 177: 745-749) was converted to the MWP P2 promoter to obtain the Brevibacillus expression vector pNK3260. It was constructed as follows. First, using pNH326 as a template, PCR was performed using two oligonucleotide primers Primer-1 and Primer-2 having the nucleotide sequences shown in SEQ ID NOs: 3 and 4, and a portion of pNH326 excluding the MWP P5 promoter was amplified. The ends were digested with restriction enzymes EcoRI and HindIII. Next, a double-stranded DNA fragment containing the MWP P2 promoter having the base sequence shown in SEQ ID NO: 5 was prepared according to a conventional method, and its ends were digested with restriction enzymes MunI and HindIII. These two DNA fragments were ligated using T4 DNA ligase to construct pNK3260.

(Example 2) Cloning of DNA sequence encoding protein A derived from Staphylococcus aureus cowan I strain (JCM2179) Staphylococcus aureus cowan I strain (JCM2179) was prepared by using T2 liquid medium (polypeptone 1%, Yeast extract 0.2%, glucose 1%, fish meat extract 0.5%, pH 7.0) and cultured with shaking at 37 ° C overnight. The cells were collected from the obtained culture broth by centrifugation, and washed twice with 10 mM Tris-HCl buffer (pH 8.0). The cells were suspended in the same buffer, lysed with 1% SDS, heated at 60 ° C. for 30 minutes, and then extracted with total methods of DNA such as phenol extraction and ethanol precipitation. Staphylococcus aureus cowan I strain (JCM2179) can be obtained from RIKEN BioResource Center, Microbial Materials Development Office (JCM) (2-1 Hirosawa, Wako, Saitama 351-0198) .

Next, based on the DNA sequence information of the protein A gene (Shuttleworth, HL et al. Gene, 1987, 58: 283-295.), Two oligonucleotide primers having the nucleotide sequences shown in SEQ ID NOs: 6 and 7 Primer-3 and Primer-4 were prepared. Using the above genomic DNA of Staphylococcus aureus cowan I strain (JCM2179) as a template, PCR is performed using these two oligonucleotide primers Primer-3 and Primer-4, and a signal sequence from protein A (S domain) A DNA fragment (about 0.9 kbp) encoding a portion excluding the cell wall binding domain (X domain) (hereinafter referred to as SPA ′) was amplified. The obtained DNA fragment was digested with restriction enzymes NcoI and BamHI, and then separated and recovered from an agarose gel.

On the other hand, the Brevibacillus expression vector pNK3260 constructed in Reference Example 1 was similarly digested with restriction enzymes NcoI and BamHI, purified and recovered, and dephosphorylated by alkaline phosphatase treatment.

After the restriction enzyme treatment, the DNA fragment encoding SPA 'and the expression vector pNK3260 were ligated using T4 DNA ligase to construct SPA' expression plasmid Spa'-pNK3260 shown in FIG. FIG. 2 shows DNA encoding the promoter, SD sequence, signal peptide and protein A (SPA ') contained in Spa'-pNK3260. The base sequence shown in SEQ ID NO: 8 shows DNA encoding a promoter, SD sequence, signal peptide and protein A (SPA ′) contained in Spa′-pNK3260, and SEQ ID NO: 9 shows signal peptide and protein A (SPA ′). ) Represents the amino acid sequence encoded by the DNA encoding.

1 and 2, “MWP-P2” is the P2 promoter region of Brevibacillus brevis cell wall protein MWP, “SDM” is the SD sequence of Brevibacillus brevis cell wall protein MWP, and “SP ′” is the Brevibacillus brevis cell wall. A modified signal peptide sequence obtained by partially modifying the signal peptide sequence of protein MWP, “spa ′” is a DNA sequence encoding SPA ′, “Nm” is a neomycin resistance gene coding region, and “Rep / pUB110” is a replica of vector pNK3260 Means the starting point. In FIG. 2, “P2-35” and “P2-10” mean the −35 region and the −10 region of the P2 promoter of Brevibacillus brevis cell wall protein MWP, respectively.

This Spa'-pNK3260 was used to transform Brevibacillus choshinensis HPD31-OK strain (FERM BP-4573) by a known method.

Comparative Example 1 Production of recombinant protein using glucose as a carbon source 1
The transformant obtained in Example 2 was treated with 3YC medium (polypeptone S3%, yeast extract 0.5%, glucose 3%, MgSO ₄ .7H ₂ O 0.01%, CaCl ₂ .7H ₂ O 0. 01%, MnSO ₄ .4H ₂ O 0.001%, FeSO ₄ .7H ₂ O 0.001%, ZnSO ₄ .7H ₂ O 0.0001% pH 7.0) to 1, 3 and 9% glucose concentration 500 ppm of Adecanol LG109 (manufactured by Adeka Co., Ltd.) was added to the prepared medium and cultured under aerobic conditions at 30 ° C. After 48 hours from the start of the culture, the culture solution is collected, the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein SPA ′ in the culture supernatant is analyzed by high performance liquid chromatography. The concentration was analyzed. As a result, the glucose concentration was 0.3 g / L at 1%, 0.9 g / L at 3%, and 0.7 g / L at 9%.

Similarly, the culture solution was collected 48 hours after the start of the culture, and the turbidity at 660 nm was analyzed using a spectrophotometer. As a result, the glucose concentration was 17 at 3%, 24 at 9%, and 16 at 9%.

(Example 3) Production of recombinant protein using fructose as a carbon source 1
The transformant obtained in Example 2 was cultured in the same manner as in Comparative Example 1 except that the carbon source of the medium was changed from glucose to fructose 1, 3, and 9%. After 48 hours from the start of the culture, the culture solution is collected, the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein SPA ′ in the culture supernatant is analyzed by high performance liquid chromatography. The concentration was analyzed. As a result, the fructose concentration was 1%, 0.6 g / L, 3%, 1.1 g / L, 9%, 0.9 g / L, which was higher than glucose added at any concentration. The results are shown in Table 1.

Similarly, 48 hours after the start of culture, the culture solution was collected and analyzed for turbidity at 660 nm using a spectrophotometer. As a result, the fructose concentration was 17% at 1%, 29 at 9%, 19 at 9%, and 19 at any concentration, which was equal to or higher than the addition of glucose. The results are shown in Table 1.

(Comparative Example 2) Production of recombinant protein using glucose as a carbon source 2
The transformant obtained in Example 2 was treated with 3YC medium (polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO ₄ .7H ₂ O 0.01%, CaCl ₂ .7H ₂ O 0. .01%, MnSO ₄ .4H ₂ O 0.001%, FeSO ₄ .7H ₂ O 0.001%, ZnSO ₄ .7H ₂ O 0.0001% pH 7.0) and Distro CC-118 (Japan Co., Ltd.) 500 ppm of fats and oils) was added and cultured under aerobic conditions at 30 ° C. After 48 hours from the start of the culture, the culture solution is collected, the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein SPA ′ in the culture supernatant is analyzed by high performance liquid chromatography. The concentration was analyzed. As a result, it was 1.3 g / L.

Similarly, the culture solution was collected 48 hours after the start of the culture, and the turbidity at 660 nm was analyzed using a spectrophotometer. As a result, it was 23.

(Example 4) Production of recombinant protein using fructose as a carbon source 2
The transformant obtained in Example 2 was treated with 3YC2 medium (peptone 1%, yeast extract 0.5%, fructose 2%, phosphate 0.3%, MgSO ₄ .7H ₂ O 0.01%, MnSO ₄ .4H ₂ O 0.001%, FeSO ₄ .7H ₂ O 0.001%, ZnSO ₄ .7H ₂ O 0.0001% pH 7.0) 750 ppm of Disperse CC-118, The cells were cultured under aerobic conditions while controlling the pH from 7.0 to 7.8. At 30 hours of culture, 2% fructose was added.

After 58 hours from the start of the culture, the culture solution is collected and the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the recombinant protein SPA ′ in the culture supernatant is measured by high performance liquid chromatography. Was 2.6 g / L.

Similarly, 10 mL of the culture solution was collected 58 hours after the start of culture, the supernatant was removed after centrifugation (3,000 rpm, room temperature, 20 minutes), and after drying sufficiently, the weight of the dried cells was analyzed. It was 10.0 g / L.

(Example 5) Production of recombinant protein using fructose as a carbon source 3
The transformant obtained in Example 2 was treated with 3YC3 medium (peptone 1%, yeast extract 0.5%, fructose 4%, phosphate 0.3%, MgSO4 · 7H2O 0.01%, MnSO4 · 4H2O. 0.001%, FeSO4 · 7H2O 0.001% , ZnSO 4 · 7H 2 O 0.0001% pH7.0, Disperse home CC 6 hours after initiation of culture to a continuous addition) of a 4% fraction fructose subjected 48 hours -118 was added at 750 ppm, and the cells were cultured under aerobic conditions of 30 ° C. while controlling the pH from 7.0 to 7.8.

After 72 hours from the start of the culture, the culture solution is collected, the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the recombinant protein SPA ′ in the culture supernatant is measured by high performance liquid chromatography. Was measured. As a result, it was 5.0 g / L, and by carrying out the culture by a novel technique, it was about 4 times as compared with 1.3 g / L in the conventional culture method using glucose shown in Comparative Example 2. It was found that the secretion amount of the recombinant protein increased. The results are shown in Table 2.

Similarly, the culture solution was collected 72 hours after the start of the culture, and the turbidity at 660 nm was analyzed using a spectrophotometer. As a result, it was 45. By culturing by a novel method, the cell density at the end of the culture was greatly doubled compared with 23 in the conventional culture method using glucose shown in Comparative Example 2 and about twice. It turned out to increase. The results are shown in Table 2.

Similarly, 10 mL of the culture solution was collected 72 hours after the start of the culture, the supernatant was removed after centrifugation (3,000 rpm, room temperature, 20 minutes), and after drying sufficiently, the dry cell weight was analyzed. . As a result, it was 15.2 g / L.

Example 6 Preparation of Transformant Expressing 5-Conjugate of Functional Variant of Protein-BR> `C-Domain Back translation was performed from an amino acid sequence (SEQ ID NO: 10, hereinafter referred to as C-G29A), and a DNA sequence encoding the protein was designed. The codon usage of the protein is close to the codon usage of HWP (J. Bacteriol., 172, p. 1312-1320, 1990), a cell surface protein that is expressed in large amounts in the Brevibacillus choshinensis HPD31 strain. The codons were distributed so that the sequence identity of the base sequences encoding each of the five domains was low. In addition, a restriction enzyme recognition site for PstI on the 5 ′ side and XbaI on the 3 ′ side of the sequence encoding the 5 linking domain was prepared. The sequence of the prepared DNA fragment is shown in SEQ ID NO: 11. The prepared DNA fragment was digested with PstI and XbaI (both manufactured by Takara), and fractionated and purified by agarose gel electrophoresis. On the other hand, pNCMO2 (manufactured by Takara), which is a plasmid vector for Brevibacillus bacteria, was purified and recovered after digestion with PstI and XbaI. After mixing both, it was ligated using Ligation High (manufactured by TOYOBO) to construct plasmid vector pNCMO2-C-G29A. Brevibacillus choshinensis SP3 strain (manufactured by Takara) was transformed using the plasmid vector obtained by the above operation.

Comparative Example 3 Production of Recombinant Protein Using Glucose as a Carbon Source 3
The transformant obtained in Example 6 was treated with 3YC medium (polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO ₄ .7H ₂ O 0.01%, CaCl ₂ .7H ₂ O 0. .01%, MnSO ₄ .4H ₂ O 0.001%, FeSO ₄ .7H ₂ O 0.001%, ZnSO ₄ .7H ₂ O 0.0001% pH 7.0) and aerobic conditions at 30 ° C. Cultured under. After 48 hours from the start of the culture, the culture solution is collected, the cells are removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein C-G29A in the culture supernatant is analyzed by high performance liquid chromatography. The concentration of was analyzed. As a result, it was 1.4 g / L.

Similarly, the culture solution was collected 48 hours after the start of the culture, and the turbidity at 660 nm was analyzed using a spectrophotometer. As a result, it was 26.

(Example 7) Production of recombinant protein using fructose as a carbon source 4
The transformant obtained in Example 6 was cultured in the same manner as in Comparative Example 2 except that the carbon source of the medium was changed from glucose to fructose 3%. After 48 hours from the start of the culture, the culture solution was collected and the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the recombinant protein C-G29A in the culture supernatant was analyzed by high performance liquid chromatography. The concentration of was analyzed. As a result, when it was cultured with glucose shown in Comparative Example 3, it was 1.4 g / L, whereas it was 2.2 g / L. The results are shown in Table 3.

Similarly, the culture solution was collected 48 hours after the start of the culture, and the turbidity at 660 nm was analyzed using a spectrophotometer. As a result, when it was cultured with glucose shown in Comparative Example 3, it was 31 compared with 26. The results are shown in Table 3.

(Example 8) From the amino acid sequence of feline proinsulin and the E and D domains of protein A shown in Preparation of transformant expressing feline proinsulin fusion protein (Non-patent Document 3), the feline proinsulin fusion protein The amino acid sequence was designed (SEQ ID NO: 12). In consideration of codon usage, a feline proinsulin fusion protein gene having the base sequence shown in SEQ ID NO: 13 was prepared. The prepared DNA fragment was digested with NcoI and EcoRI (both manufactured by Takara), and fractionated and purified by agarose gel electrophoresis. On the other hand, pNH326, which is a plasmid vector for Brevibacillus spp., Was digested with NcoI and EcoRI and purified and recovered. After mixing both, it was ligated using Ligation High (manufactured by TOYOBO) to construct a plasmid vector pNH326EDCIP. Using the plasmid vector obtained by the above operation, a transformant of Brevibacillus choshinensis HPD31-OK was prepared.

Comparative Example 4 Production of recombinant protein using glucose as a carbon source 4
The transformant obtained in Example 8 was treated with 3YC medium (polypeptone S 3%, yeast extract 0.5%, glucose 3%, MgSO ₄ .7H ₂ O 0.01%, CaCl ₂ .7H ₂ O 0. 0.01%, MnSO ₄ .4H ₂ O 0.001%, FeSO ₄ .7H ₂ O 0.001%, ZnSO ₄ .7H ₂ O 0.0001% pH 7.0) The cells were cultured under aerobic conditions at 30 ° C. After 48 hours from the start of the culture, the culture solution was collected and the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the feline proinsulin fusion protein in the culture supernatant by SDS-PAGE. Was analyzed.

(Example 9) Production of recombinant protein using fructose as a carbon source 5
The transformant obtained in Example 8 was cultured in the same manner as in Comparative Example 3, except that the carbon source of the medium was changed from glucose to fructose 3%. After 48 hours from the start of the culture, the culture solution was collected and the cells were removed by centrifugation (10,000 rpm, 4 ° C., 5 minutes), and then the concentration of the feline proinsulin fusion protein in the culture supernatant by SDS-PAGE. Was analyzed by ChemiDoc XRS system (Bio-Rad). As a result, about twice the production amount when cultured with glucose shown in Comparative Example 4 was confirmed. The results are shown in Table 4.

From the above results, it has been clarified that the production of recombinant protein of the genus recombinant Brevibacillus using the fructose of the present invention as a carbon source can improve the productivity of the recombinant protein. Furthermore, with regard to protein A, by using fructose as a carbon source and appropriately selecting the addition amount and addition method of fructose, it has been possible to achieve a productivity that has been reported to be about 4 times, which is a problem in the past. It became clear that the problem of low productivity could be solved.

Claims (11)

Production of a recombinant protein using a recombinant Brevibacillus bacterium, wherein the recombinant protein is cultured using fructose as a carbon source, and the initial concentration of fructose is 1% or more. Method. The method for producing a recombinant protein according to claim 1, wherein the initial concentration of fructose is 9% or less. The method for producing a recombinant protein according to claim 1 or 2, wherein the fructose concentration is additionally added so as to be 9% or less. The method for producing a recombinant protein according to claim 3, wherein the fructose concentration is additionally added to 4% or less. The method for producing a recombinant protein according to claim 3 or 4, wherein the additional addition method of fructose is intermittent or continuous. The method for producing a recombinant protein according to any one of claims 1 to 5, wherein the recombinant protein is an antibody-binding protein. The antibody-binding protein is one or more proteins selected from the group consisting of protein A, protein G, protein L, their partial sequences having antibody binding activity, functional variants thereof, and conjugates thereof. A method for producing a recombinant protein according to claim 6. The method for producing a recombinant protein according to any one of claims 1 to 5, wherein the recombinant protein is a physiologically active protein derivative. The method for producing a recombinant protein according to claim 8, wherein the physiologically active protein is a peptide hormone or a precursor thereof. The method for producing a recombinant protein according to claim 9, wherein the peptide hormone or a precursor thereof is insulin, proinsulin, or preproinsulin. The method for producing a recombinant protein according to claim 10, wherein the insulin, proinsulin, or preproinsulin is feline insulin, feline proinsulin, or feline preproinsulin.

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