Method for producing protein of interest

Abstract

Provided is a method for producing a protein of interest using a gram-positive bacterium with improved productivity. The method for producing a protein of interest includes culturing a gram-positive bacterium containing a gene encoding a heterologous FK506-binding protein (FKBP) and a gene encoding the protein of interest, or mixed-culturing a gram-positive bacterium containing a gene encoding a heterologous FKBP and a gram-positive bacterium containing a gene encoding the protein of interest.

Description

Method for producing target protein

[0001] The present invention relates to a method for producing a target protein with improved productivity.

[0002] The industrial production of useful substances by microorganisms covers a wide range of fields, including foods, amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, and proteins, and their uses are also widespread, ranging from everyday items such as foods, medicines, detergents, and cosmetics, to raw materials for various chemical products. Industrially useful host microorganisms include Escherichia coli, Bacillus subtilis, yeast, and filamentous fungi. In the industrial production of useful substances by such microorganisms, improving productivity is one of the important challenges.

[0003] Proline isomerase (PPIase) catalyzes the isomerization of prolyl bonds and can assist in protein folding (Non-Patent Document 1). PPIases consist of three families: cyclophilins, FK506-binding proteins (FKBPs), and parvulins, which differ in their sensitivity to inhibitors. It is known that the amino acid sequence identity between the three PPIase families is low. PPIases from all three families are present in the periplasm of Escherichia coli. Among these, FkpA, a member of the FKBP family, has been extensively studied, and it has been reported that increasing its expression level can improve target protein productivity (Non-Patent Document 2). It has also been reported that the N-terminal chaperone region of FkpA contributes to improved target protein productivity in E. coli (Non-Patent Document 3). It has been reported that the productivity of target proteins can be improved both in cell-free systems and by adding any of the three families of PPIases (Patent Document 1).In the secretory production of target proteins in Gram-positive bacteria, the enhancement of expression of parvulin, including PrsA, has been extensively studied (Non-Patent Document 4).

[0004] The Gram-positive bacterium Bacillus subtilis has PrsA as an endogenous chaperone and PPIase (Non-Patent Document 5), and it has been reported that enhancing the expression level of PrsA improves the productivity of the target protein (Patent Document 2, Non-Patent Documents 6 and 7). PrsA is anchored to the outer surface of the cell membrane and promotes the folding of proteins transported via translocase. Since improperly folded proteins are subject to degradation by proteases present in the cell wall and at the cell membrane-cell membrane interface, they must be correctly folded immediately after the signal peptide is cleaved (Non-Patent Document 4).

[0005] On the other hand, it has not been reported so far what effect FKBP has on the production of target proteins in Gram-positive bacteria.

[0006] (Patent Document 1) Japanese Patent Application Publication No. 2005-253432 (Patent Document 2) Patent No. 4202985 (Non-Patent Document 1) Journal of Molecular Biology, 2015, 427(7): 1609-1631 (Non-Patent Document 2) Biotechnol Prog. 2017, 33(1): 212-220 (Non-patent document 3) Microbial Cell Factories, 2010, volume 9, Article number: 22 (Non-patent document 4) Microbial Cell Factories, 2019, volume 18, Article number: 158 (Non-patent document 5) J Biol Chem. 2004, 279(18):19302-14 (Non-patent document 6) Journal of Bacteriology, 1998, 180(11): 2830-2835 (Non-patent document 7) Journal of Bacteriology, 2001, 183(6): 1881-1890

[0007] The present invention relates to the following 1) and 2): 1) A method for producing a target protein, comprising culturing a Gram-positive bacterium containing a gene encoding a heterologous FKBP and a gene encoding a target protein, or co-culturing a Gram-positive bacterium containing a gene encoding a heterologous FKBP and a Gram-positive bacterium containing a gene encoding a target protein; and 2) a Gram-positive bacterium containing a gene encoding a heterologous FKBP, or a gene encoding a heterologous FKBP and a gene encoding a target protein.

[0008] Fab production amount when each PPIase is expressed in a Fab-producing Bacillus subtilis strain. Fab production amount when PrsA or FkpA is expressed in a Fab-producing Bacillus subtilis strain. Fab production amount when the entire region of FkpA (FkpA), the N-terminal region (FkpA_N), or the C-terminal region (FkpA_C) is expressed in a Fab-producing Bacillus subtilis strain. Fab production amount when FkpA is secreted and expressed (FkpA_p) or when it is expressed anchored to the cell membrane (FkpA_m) in a Fab-producing Bacillus subtilis strain. Phylogenetic tree of FKBP. HMM profile of the BCFK clade. Detailed Description of the Invention

[0009] As used herein, the identity of a nucleotide sequence or an amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, the identity is calculated by performing an analysis using the Search homology program in the genetic information processing software Genetyx-Win, with the unit size to compare (ktup) set to 2.

[0010] As used herein, "at least 80% identity" with respect to an amino acid sequence or a nucleotide sequence refers to identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0011] As used herein, a "corresponding position" or "corresponding region" in an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to maximize homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Nucleic Acids Res, 1994, 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 and Clustal omega, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the Clustal website (www.clustal.org) operated by University College Dublin, the European Bioinformatics Institute (EBI) (www.ebi.ac.uk / index.html), and the DNA Data Bank of Japan (DDBJ) (www.ddbj.nig.ac.jp / searches-j.html) operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the above-described alignment is considered to be the "position corresponding to" that position. Furthermore, the region flanked by corresponding positions or the region consisting of a corresponding motif is also considered to be the corresponding region.

[0012] Those skilled in the art can further fine-tune the alignment of amino acid sequences obtained above to optimize it. Such optimal alignment is preferably determined taking into account the identity or similarity of the amino acid sequences, the frequency of inserted gaps, and the like. Here, the identity or similarity of amino acid sequences refers to the percentage (%) of the number of positions where identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned, relative to the total number of amino acid residues. Similar amino acid residues refer to amino acid residues among the 20 amino acids that constitute proteins that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Such groups of similar amino acid residues are well known to those skilled in the art, and include, but are not limited to, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; leucine and isoleucine.

[0013] As used herein, the term "amino acid residue" refers to the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0014] As used herein, the phrase "operably linked" between a regulatory region and a gene means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene and a regulatory region are well known to those skilled in the art.

[0015] As used herein, "upstream" and "downstream" in relation to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the 5' region of the gene on the DNA sense strand.

[0016] The names of the Bacillus subtilis genes described herein are based on the Bacillus subtilis genome data published on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) ([bacillus.genome.ad.jp / ], updated January 18, 2006). The Bacillus subtilis gene numbers described herein refer to the gene numbers registered in the BSORF DB.

[0017] As used herein, FK506-binding protein (FKBP) refers to an enzyme that is a member of the family of peptidylprolyl isomerases (PPIases, EC 5.1.2.8) that catalyze the isomerization of prolyl bonds and can assist protein folding. FKBP is a group of PPIase enzymes whose activity is inhibited by binding to FK506, an immunosuppressant.

[0018] As used herein, a "clade" is a grouping of sequences from a common ancestor and all descendants of that common ancestor (evolution.berkeley.edu / evolibrary / article / 0_0_0 / evo_06). Clades can be visualized as phylogenetic trees and share common characteristics. Subclades that group together within a clade in a phylogenetic tree can also share common characteristics, and sequences in one subclade are more closely related to each other than sequences in other subclades within that clade.

[0019] As used herein, the term "BCFK clade" refers to a group of sequences belonging to the FKBP family. Sequences belonging to the BCFK clade are defined as sequences having an HMM score of 115 or greater across the entire length of their amino acid sequences against the Hidden Markov Model (HMM) profile shown in Figure 6. Preferred sequences belonging to the BCFK clade are sequences having an HMM score of 150 or greater across the entire length of their amino acid sequences against the HMM profile shown in Figure 6. The HMM profile in Figure 6 was created by constructing a multiple alignment using MAFFT v7.471 with FKBP sequences belonging to the BCFK clade, and then using HMMER version 3.3.2 (http: / / hmmer.org / ) with the parameters --pnone and --wnone. The Uniprot numbers of the FKBP sequences used to create the HMM profile defining the BCFK clade are shown in Table 3 below. By binarizing the HMM profile using hmmpress and performing a homology search using hmmscan, the HMM score of any sequence against the created HMM profile of the BCFK clade can be calculated.

[0020] As used herein, the term "antibody-related molecule" refers to an immunoglobulin or a protein containing a molecular species consisting of a single domain selected from the domains constituting immunoglobulins or a combination of two or more domains. Examples of domains constituting immunoglobulins include VH, CH1, CH2, and CH3, which are domains of immunoglobulin heavy chains, and VL and CL, which are domains of immunoglobulin light chains. An antibody-related molecule may be a monomeric protein or a multimeric protein. When an antibody-related molecule is a multimeric protein, it may be a homomultimer consisting of a single type of subunit, or a heteromultimer consisting of two or more types of subunits. Preferably, an antibody-related molecule is a molecule that contains an immunoglobulin antigen-recognition domain and can specifically bind to a target such as a protein. Examples of antibody-related molecules include immunoglobulins such as IgG, as well as Fab and F(ab') ２Examples of such antibodies include small molecular weight antibodies, such as single chain antibodies (scFv), diabodies, and variable domains of heavy chains of heavy chain antibodies (VHH).

[0021] As used herein, the term "Fab" refers to an antibody consisting of the VH-CH1 domain of an immunoglobulin heavy chain and the VL-CL domain of an immunoglobulin light chain.

[0022] As used herein, "VHH" refers to the heavy chain variable domain of a heavy chain antibody. Heavy chain antibodies have been found in camelids and cartilaginous fish such as sharks. Those derived from cartilaginous fish such as sharks are also called VNAR (single variable new antigen receptor domain antibodies). The VHH used herein is derived from, for example, a camelid or shark, preferably a camelid. A VHH is a molecule capable of recognizing an antigen with a single domain and is the smallest unit of antibody molecules discovered to date. The VHH produced according to the present invention can contain one or more heavy chain variable domains derived from a heavy chain antibody, and the number of heavy chain variable domains contained in the VHH is not limited. The heavy chain variable domain contained in the VHH produced according to the present invention may be a heavy chain variable domain derived from a natural heavy chain antibody or a modified version thereof. Furthermore, the VHH produced according to the present invention may contain fragments other than the heavy chain variable domain derived from the immunoglobulin heavy chain (e.g., fragments of the constant region of a heavy chain antibody, or human-derived fragments, linker sequences, etc.), or amino acid mutations for stabilization.

[0023] In this specification, the Camelidae includes Bactrian camels, dromedaries, llamas, alpacas, vicunas, guanacos, etc., and preferably includes alpacas.

[0024] The present invention relates to providing a method for producing a target protein using Gram-positive bacteria, typically Bacillus subtilis, with improved productivity.

[0025] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that in the production of a target protein using Gram-positive bacteria, the productivity of the target protein can be improved by causing the Gram-positive bacteria to express a specific PPIase derived from a heterologous organism.

[0026] According to the present invention, it becomes possible to efficiently produce a target protein using Gram-positive bacteria, such as Bacillus subtilis.

[0027] The method for producing a target protein of the present invention comprises culturing a Gram-positive bacterium containing a gene encoding a heterologous FKBP and a gene encoding a target protein, or co-culturing a Gram-positive bacterium containing a gene encoding a heterologous FKBP and a Gram-positive bacterium containing a gene encoding a target protein.

[0028] In the method of the present invention, the FKBP is not particularly limited as long as it is a heterologous FKBP. Here, "heterologous" refers to a microorganism or organism classified as a species other than that to which the Gram-positive bacterium used to produce the target protein belongs. From the viewpoint of improving productivity of the target protein, such heterologous FKBP is preferably a polypeptide belonging to the BCFK clade and having prolyl bond isomerization activity, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 6 and having prolyl bond isomerization activity, and more preferably a polypeptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence set forth in any of SEQ ID NOs: 1 to 6 and having prolyl bond isomerization activity.

[0029] The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 is MJFKS derived from Methanococcus jannaschii. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is FkpA derived from Escherichia coli. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 is FKBP_1 derived from Lelliottia amnigena. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 is FKBP_K derived from Klebsiella quasipneumoniae. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 is FKBP_E derived from Erwinia sp. B116. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 5 is FKBP_P derived from Pantoea sp. Nvir. The polypeptide consisting of any of the amino acid sequences set forth in SEQ ID NOs: 1 to 6 is a polypeptide belonging to the FKBP family and has prolyl bond isomerization activity. Of these, the polypeptide consisting of any of the amino acid sequences set forth in SEQ ID NOs: 1 to 5 is a polypeptide belonging to the BCFK clade.

[0030] "Prolyl bond isomerization activity" refers to the activity of isomerizing cis-trans prolyl bonds in a protein. Prolyl bond isomerization activity can be evaluated using methods well known in the art. For example, the activity can be determined by measuring the refolding rate of ribonuclease T1 (RNase) by the polypeptide of interest (Marika Vitikainen et al. Journal of Biological Chemistry, 2004, 279(18): 19302-19314).

[0031] The gene encoding a heterologous FKBP is a polynucleotide encoding the heterologous FKBP described above. Examples of genes encoding heterologous FKBP include a polynucleotide encoding FkpA consisting of the nucleotide sequence shown in SEQ ID NO: 35, a polynucleotide encoding FKBP_1 consisting of the nucleotide sequence shown in SEQ ID NO: 40, a polynucleotide encoding FKBP_K consisting of the nucleotide sequence shown in SEQ ID NO: 41, a polynucleotide encoding FKBP_E consisting of the nucleotide sequence shown in SEQ ID NO: 42, a polynucleotide encoding FKBP_P consisting of the nucleotide sequence shown in SEQ ID NO: 43, a polynucleotide encoding MJFKS, and a polynucleotide having at least 80% identity to any of the nucleotide sequences shown in SEQ ID NOs: 35, 40 to 43, and 46 and encoding a polypeptide having prolyl bond isomerization activity, and a polynucleotide consisting of any of the nucleotide sequences shown in SEQ ID NOs: 35, 40 to 43, and 46 is more preferred.

[0032] A gene encoding a heterologous FKBP can be prepared according to conventional methods. For example, a gene encoding a heterologous FKBP can be prepared by extracting genomic DNA from a microorganism or organism that originally produces the heterologous FKBP, or by extracting RNA and synthesizing cDNA by reverse transcription, according to conventional methods. For example, a gene encoding FkpA (SEQ ID NO: 35) can be prepared from Escherichia coli (NBRC 3301) or the like. The above microorganisms can be purchased from public microorganism collections. A gene encoding a mutated heterologous FKBP may be prepared by further site-directed mutagenesis of the gene encoding the heterologous FKBP obtained by the above procedure. Alternatively, a gene encoding a heterologous FKBP may be chemically synthesized based on the amino acid sequence of the heterologous FKBP. Furthermore, a gene encoding a heterologous FKBP may be codon-optimized to suit the species of Gram-positive bacteria used as the host cell. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0033] The gene encoding the heterologous FKBP may be operably linked to a regulatory region. A "regulatory region" is a region that has the function of controlling the intracellular expression of a gene located downstream thereof, and preferably has the function of constitutively expressing or highly expressing the gene located downstream thereof. More specifically, it can be defined as a region that is located upstream of the coding region of a gene and has the function of interacting with RNA polymerase to control the transcription of the coding region. Preferably, the regulatory region in this specification refers to a region approximately 200 to 600 nucleotides upstream of the coding region of a gene. The regulatory region includes a transcription initiation regulatory region and / or a translation initiation regulatory region, or the region from the transcription initiation regulatory region to the translation initiation regulatory region. The transcription initiation control region is a region including a promoter and a transcription initiation site, and the translation initiation control region is a site corresponding to the Shine-Dalgarno (SD) sequence which forms a ribosome binding site together with an initiation codon (Shine, J., Dalgarno, L., Proc. Natl. Acad. Sci. USA., 1974, 71: 1342-1346).

[0034] Preferred examples of the regulatory region include, but are not particularly limited to, regulatory regions that function in Gram-positive bacteria, preferably bacteria of the genus Bacillus, for example, the regulatory regions of the α-amylase gene, protease gene, aprE gene, or spoVG gene derived from bacteria of the genus Bacillus, the regulatory region of the cellulase gene of Bacillus sp. KSM-S237 strain (JP 2000-210081 A), the regulatory region of the cellulase gene of Bacillus sp. KSM-64 strain (JP 2011-10387 A), and the regulatory region of the kanamycin resistance gene or chloramphenicol resistance gene derived from Staphylococcus aureus (see JP 2009-089708 A for both). More preferred examples of the regulatory region include promoters derived from bacteria of the genus Bacillus, such as the promoter of the cellulase gene of Bacillus sp. KSM-S237 strain (SEQ ID NO: 94), the promoter of the cellulase gene of Bacillus sp. KSM-64 strain (SEQ ID NO: 95), and the promoter of the spoVG gene of Bacillus subtilis (SEQ ID NO: 96). Further preferred regulatory regions include promoters consisting of a nucleotide sequence having at least 90% identity to any of SEQ ID NOs: 94 to 96.

[0035] Furthermore, from the viewpoint of improving productivity of the target protein, it is preferable that the gene encoding the heterologous FKBP be operably linked to a polynucleotide encoding a secretory signal peptide (referred to as a secretory signal sequence) that functions to secrete the expressed FKBP extracellularly. Preferred examples of secretory signal sequences include secretory signal sequences that function in Gram-positive bacteria, preferably bacteria of the genus Bacillus, such as secretory signal sequences derived from bacteria of the genus Bacillus. Preferred examples of secretory signal sequences derived from bacteria of the genus Bacillus include the secretory signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain (SEQ ID NO: 97), the secretory signal sequence of the cellulase gene of Bacillus sp. KSM-64 strain (SEQ ID NO: 98), and the secretory signal sequence of the amylase gene amyE of Bacillus subtilis (SEQ ID NO: 99). Further examples of secretory signal sequences derived from bacteria of the genus Bacillus include nucleotide sequences that share at least 80% identity with any of SEQ ID NOs: 97 to 99 and have the function of secreting the expressed protein extracellularly. The sequence encoding the heterologous FKBP linked to the secretory signal sequence derived from these Bacillus bacteria may or may not contain the secretory signal sequence of the native heterologous FKBP.

[0036] Thus, a gene encoding a heterologous FKBP may contain nucleotide sequences of untranslated regions (UTRs) in addition to the open reading frame (ORF). For example, the gene may contain the promoter, secretory signal sequence, and terminator described above.

[0037] In the method of the present invention, the gene encoding the target protein may be a foreign gene (heterologous gene) encoding a heterologous protein, a gene encoding a protein derived from the same species introduced from outside, or an endogenous gene encoding a protein that the host cell can naturally express. The target protein is preferably a protein having at least one prolyl bond isomerization site. Here, the prolyl bond isomerization site in the target protein refers to a site that can be isomerized during the production process of the target protein, i.e., a cis-prolyl bond site. The presence and / or number of cis-prolyl bond sites in the target protein can be confirmed by referring to the three-dimensional structure of the target protein obtained by X-ray crystal structure analysis. The type of target protein is not particularly limited. Examples of target proteins include enzymes involved in the synthesis of desired proteins or desired target substances, such as antibody-related molecules, cytokines, hormones, other physiologically active peptides, transporters, industrial enzymes such as proteases, and metabolic enzymes.

[0038] Preferably, the target protein is an antibody-related molecule, more preferably a small molecule antibody such as Fab, ScFv, or VHH. Even more preferably, the target protein is selected from the group consisting of Fab, ScFv, and VHH, and even more preferably Fab.

[0039] A gene encoding a target protein can be prepared according to conventional methods. For example, a gene encoding a target protein can be prepared by extracting genomic DNA from a microorganism or organism that originally produces the target protein, or by extracting RNA and synthesizing cDNA by reverse transcription, according to conventional methods. Alternatively, a gene encoding a target protein can be chemically synthesized based on the amino acid sequence of the target protein. Furthermore, a gene encoding a target protein may be codon-optimized to suit the species of Gram-positive bacteria used as the host cell.

[0040] The gene encoding the target protein may be operably linked to a regulatory region. Preferably, the gene encoding the target protein is operably linked to a secretory signal sequence that causes the expressed target protein to be secreted outside the cell. Details of the regulatory region and secretory signal are the same as those described for the gene encoding the heterologous FKBP.

[0041] The gene encoding a heterologous FKBP and the gene encoding a target protein may be introduced into the same Gram-positive bacterium, or may be introduced into different Gram-positive bacteria. When the gene encoding a heterologous FKBP and the gene encoding a target protein are introduced into different Gram-positive bacteria, the Gram-positive bacteria may be of the same species or different species, but from the viewpoint of culture efficiency, the Gram-positive bacteria are preferably of the same species. Furthermore, when the gene encoding a heterologous FKBP and the gene encoding a target protein are introduced into different Gram-positive bacteria, it is preferable that each gene is operably linked to a secretion signal sequence. More preferably, the gene encoding a heterologous FKBP and the gene encoding a target protein are introduced into the same Gram-positive bacterium.

[0042] The Gram-positive bacterium used as a host cell is not particularly limited as long as it can be used for protein production, and examples thereof include Bacillus bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, Bacillus thuringiensis, and Bacillus amyloliquefaciens, and Clostridium bacteria such as Clostridium butyricum. Of these, Bacillus bacteria are preferred, and Bacillus subtilis is more preferred.

[0043] The Gram-positive bacterium may be a wild-type strain or a mutant strain that has been mutated. For example, a Bacillus subtilis mutant strain that is deficient in at least one extracellular protease gene is preferably used to effectively prevent extracellular degradation of proteins secreted outside the cells and thereby improve protein productivity. Here, "deficient" in an extracellular protease means that the activity of the extracellular protease is reduced compared to the activity of a cell that is not deficient in the extracellular protease (e.g., a wild-type strain). Cells deficient in an extracellular protease (extracellular protease-deficient strain) can be produced by partial or complete deletion or inactivation (so-called knockdown or knockout) of the gene encoding the extracellular protease in the cell. Preferably, the extracellular protease-deficient strain has an extracellular protease activity that is reduced to 50% or less, more preferably 25% or less, compared to that of a wild-type strain. The extracellular protease gene deleted in the extracellular protease-deficient Bacillus subtilis strain is preferably at least one gene, more preferably three genes, and even more preferably all genes selected from the group consisting of aprE (BSORF DB gene number: BG10190), epr (BG10561), wprA (BG11846), mpr (BG10690), nprB (BG10691), bpr (BG10233), nprE (BG10448), vpr (BG10591), and aprX (BG12567). A preferred example of such a Bacillus subtilis mutant strain is the extracellular protease-deficient Bacillus subtilis strain Dpr9 (see Japanese Patent No. 4,485,341). Furthermore, the Bacillus subtilis mutant strain may be subjected to deletion of a sigma factor such as sigF (see Japanese Patent No. 4336082), deletion of recA involved in homologous recombination activity (see Japanese Patent No. 6088282), etc. A Bacillus subtilis mutant strain obtained by adding deletion of one or both of sigF and recA to the Dpr9 strain can also be used.

[0044] Introduction of a gene encoding a heterologous FKBP, a gene encoding a target protein, or a gene encoding a heterologous FKBP and a gene encoding a target protein into a Gram-positive bacterium host cell can be carried out according to standard methods. For example, a gene encoding a heterologous FKBP can be integrated into the genome of a host Gram-positive bacterial cell by introducing a gene encoding a heterologous FKBP or a vector containing the gene into the host Gram-positive bacterial cell. A gene encoding a target protein can be integrated into the genome of a host Gram-positive bacterial cell by introducing a gene encoding a target protein or a vector containing the gene into the host Gram-positive bacterial cell. A gene encoding a heterologous FKBP and a gene encoding a target protein can be integrated into the genome of a host Gram-positive bacterial cell by introducing a gene encoding a heterologous FKBP or a vector containing the gene and a gene encoding a target protein or a vector containing the gene, or a vector containing both a gene encoding a heterologous FKBP and a gene encoding a target protein into the host Gram-positive bacterial cell. Alternatively, an expression vector containing a gene encoding a heterologous FKBP, an expression vector containing a gene encoding a target protein, an expression vector containing a gene encoding a heterologous FKBP and a gene encoding a target protein, or an expression vector containing both a gene encoding a heterologous FKBP and a gene encoding a target protein may be introduced into a host Gram-positive bacterial cell. When two types of genes or vectors are introduced into the same host cell, the order of introduction is not limited, and either may be introduced first, or they may be introduced simultaneously.

[0045] To introduce a gene or vector into Gram-positive bacterial cells, known transformation techniques such as the competent cell method, electroporation, protoplast method, particle gun method, and PEG method can be applied.

[0046] A vector containing a gene encoding a heterologous FKBP, a vector containing a gene encoding a target protein, or a vector containing a gene encoding a heterologous FKBP and a gene encoding a target protein can be constructed by inserting and ligating the gene encoding the heterologous FKBP and / or the gene encoding the target protein, and, if necessary, a regulatory region or secretory signal sequence, into any vector using conventional methods. The type of vector is not particularly limited, and may be any vector such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, or shuttle vector. Furthermore, the vector is preferably a vector that can be amplified in host cells, and more preferably an expression vector. Preferred examples of the vector include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R, or pASP64 (Japanese Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60: 235-243), pHY-S237 (Japanese Patent Laid-Open No. 2014-158430), and pAC3 (Nucleic Acids Res, 1988, 16: 8732); and plasmids usable for transformation of Bacillus bacteria such as pUB110 (J Bacteriol, 1978, 134: 318-329) and pTA10607 (Plasmid, 1987, 18: 8-15). Plasmids derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0047] In the method of the present invention, Gram-positive bacteria containing a gene encoding a heterologous FKBP and a gene encoding a target protein, obtained by the above-described procedure, are cultured, or Gram-positive bacteria containing a gene encoding a heterologous FKBP and a gene encoding a target protein are co-cultured. The Gram-positive bacteria may be cultured according to a typical Gram-positive bacterial culture method. For example, a medium for culturing Gram-positive bacteria contains carbon and nitrogen sources necessary for bacterial growth. Carbon sources include glucose, dextran, soluble starch, sucrose, and methanol. Nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. If necessary, the medium may contain other nutrients, such as inorganic salts (e.g., sodium chloride, calcium chloride, sodium dihydrogen phosphate, and magnesium chloride), vitamins, and antibiotics (e.g., tetracycline, neomycin, kanamycin, spectinomycin, and erythromycin). Culture conditions, such as temperature, aeration and agitation conditions, pH of the medium, and culture time, can be appropriately selected depending on the bacterial species, characteristics, culture scale, etc.

[0048] By culturing the Gram-positive bacterium, the target protein is expressed intracellularly. Furthermore, if the gene encoding the target protein is linked to a polynucleotide encoding a secretory signal peptide, the expressed target protein is secreted extracellularly. The target protein produced by the method of the present invention can be recovered from the culture medium by a common protein purification method, such as cell disruption, centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in combination.

[0049] According to the method of the present invention, as shown in the Examples below, expression of a heterologous FKBP during expression of a target protein in Gram-positive bacteria significantly improves target protein productivity compared to expression of the target protein alone. Furthermore, target protein productivity is also significantly improved compared to expression of a target protein in Gram-positive bacteria in the coexistence of PrsA, which has been reported to contribute to improved target protein productivity in Bacillus subtilis (see Patent Document 2, Non-Patent Documents 5 and 6). Thus, the method of the present invention enables efficient production of a target protein with high productivity using Gram-positive bacteria, thereby reducing the time and cost required for target protein production.

[0050] Furthermore, by secreting and expressing a heterologous FKBP during expression of a target protein in Gram-positive bacteria, productivity of the target protein is significantly improved compared to when the heterologous FKBP is expressed in a state where it is anchored to the cell membrane. Given that proteins produced in Bacillus subtilis are known to fold near the cell membrane (see Non-Patent Document 4), it was completely unexpected that the productivity of a target protein is improved when an FKBP capable of assisting protein folding is secreted rather than expressed near the cell membrane.

[0051] The present invention also encompasses the following substances, manufacturing methods, uses, methods, etc. as exemplary embodiments, but the present invention is not limited to these embodiments.

[0052] [1] A method for producing a target protein, comprising culturing Gram-positive bacteria containing a gene encoding a heterologous FKBP and a gene encoding a target protein, or co-culturing Gram-positive bacteria containing a gene encoding a heterologous FKBP and a Gram-positive bacterium containing a gene encoding a target protein, preferably culturing Gram-positive bacteria containing a gene encoding a heterologous FKBP and a gene encoding a target protein. [2] A method for improving productivity of a target protein, comprising culturing Gram-positive bacteria containing a gene encoding a heterologous FKBP and a gene encoding a target protein, or co-culturing Gram-positive bacteria containing a gene encoding a heterologous FKBP and a Gram-positive bacterium containing a gene encoding a heterologous FKBP and a gene encoding a target protein, preferably culturing Gram-positive bacteria containing a gene encoding a heterologous FKBP and a gene encoding a target protein. [3] The method of [1] or [2], wherein the heterologous FKBP is a polypeptide belonging to the BCFK clade and having prolyl bond isomerization activity, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 6 and having prolyl bond isomerization activity, preferably a polypeptide belonging to the BCFK clade and having prolyl bond isomerization activity or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6. [4] The method of [1] or [2], wherein the heterologous FKBP is a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in any of SEQ ID NOs: 1 to 6 and having prolyl bond isomerization activity, preferably a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 6.[5] The method of [1] or [2], wherein the gene encoding the heterologous FKBP is a polynucleotide consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 35, 40 to 43, and 46, or a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 35, 40 to 43, and 46 and encoding a polypeptide having prolyl bond isomerization activity, preferably a polynucleotide consisting of the nucleotide sequence set forth in any one of SEQ ID NOs: 35, 40 to 43, and 46. [6] The method of any one of [1] to [5], wherein the gene encoding the heterologous FKBP is operably linked to a polynucleotide encoding a secretory signal peptide. [7] The method of any one of [1] to [6], wherein the Gram-positive bacterium is a bacterium of the genus Bacillus, preferably Bacillus subtilis or a mutant thereof. [8] The method according to any one of [1] to [6], wherein the Gram-positive bacterium is Bacillus subtilis in which at least one extracellular protease gene has been deleted or inactivated, preferably Bacillus subtilis in which at least one extracellular protease gene selected from the group consisting of aprE, epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX has been deleted or inactivated, more preferably Bacillus subtilis in which aprE, epr, wprA, mpr, nprB, bpr, nprE, vpr, and aprX have been deleted or inactivated. [9] The method according to any one of [1] to [8], wherein the target protein has at least one prolyl bond isomerization site.

[10] The method according to any one of [1] to [9], wherein the target protein is an antibody-related molecule, preferably selected from the group consisting of Fab, ScFv, and VHH, and more preferably Fab.

[11] The method according to any one of [1] to

[10] , further comprising recovering the target protein from the culture.

[0053]

[12] A Gram-positive bacterium comprising a gene encoding a heterologous FKBP, or a gene encoding a heterologous FKBP and a gene encoding a target protein.

[13] The Gram-positive bacterium according to

[12] , wherein the heterologous FKBP is a polypeptide belonging to the BCFK clade and having prolyl bond isomerization activity, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 6 and having prolyl bond isomerization activity, preferably a polypeptide belonging to the BCFK clade and having prolyl bond isomerization activity or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6.

[14] The Gram-positive bacterium according to

[12] , wherein the heterologous FKBP is a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in any of SEQ ID NOs: 1 to 6 and having prolyl bond isomerization activity, preferably a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 6.

[15] The Gram-positive bacterium according to

[12] , wherein the gene encoding the heterologous FKBP is a polynucleotide consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 35, 40 to 43, and 46, or a polynucleotide consisting of a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 35, 40 to 43, and 46 and encoding a polypeptide having prolyl bond isomerization activity, preferably a polynucleotide consisting of the nucleotide sequence shown in any one of SEQ ID NOs: 35, 40 to 43, and 46.

[16] The Gram-positive bacterium according to any one of

[12] to

[15] , wherein the gene encoding the heterologous FKBP is operably linked to a polynucleotide encoding a secretory signal peptide.

[17] The Gram-positive bacterium according to any one of

[12] to

[16] , which is a bacterium of the genus Bacillus, preferably Bacillus subtilis or a mutant thereof.

[18] The Gram-positive bacterium according to any one of

[12] to

[16] , wherein the target protein has at least one prolyl bond isomerization site.

[20] The Gram-positive bacterium according to any one of

[12] to

[19] , wherein the target protein is an antibody-related molecule, preferably selected from the group consisting of Fab, ScFv, and VHH, and more preferably Fab.

[0054] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0055] Materials and Methods (1) Host Strain The host Bacillus subtilis used was a derivative of Bacillus subtilis strain 168. A sigF gene-deficient strain (Dpr9ΔsigF) was constructed from the extracellular protease-deficient Bacillus subtilis strain Dpr9 (see Japanese Patent No. 4485341, deficient in extracellular proteases epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX) according to the method described in Japanese Patent No. 4336082.

[0056] (2) Culture medium LB medium: 1% Bacto ＴＭ Tryptone, 0.5% Bacto ＴＭ Yeast extract, 1% sodium chloride. 1.5% agar was added to the plate medium. Tetracycline (50 ppm) was added as needed. DM3 medium: 1% CMC (Kanto Chemical), 0.5% Bacto ＴＭ Casamino Acids, 0.5% Bacto ＴＭ Yeast Extract, 8.1% Disodium Succinate 6H２ 0, 0.35% dipotassium hydrogen phosphate, 0.15% dipotassium hydrogen phosphate, 0.5% glucose, 20 mM magnesium chloride, 0.01% BSA, 50 ppm tetracycline. 1% agar was added to the plate medium. 2x L-mal medium: 2% Bacto ＴＭ Tryptone, 1% Bacto ＴＭ Yeast Extract, 1% sodium chloride, 7.5% maltose monohydrate, 7.5 ppm manganese sulfate, 15 ppm tetracycline.

[0057] Example 1: Enhanced Protein Production by PPIase Expression 1-1. Construction of a Plasmid for PPIase Expression Using the recombinant plasmid pHY-S237 (JP 2014-158430 A), which was constructed based on pHY300PLK, as a template, the plasmid sequence was amplified by PCR using the primer set of SEQ ID NO:7 and SEQ ID NO:8 and PrimeSTAR Max DNA polymerase (TaKaRa). Using the genome of strain 168 as a template, promoter DNA derived from the spoVG gene was amplified by PCR using the primer set of SEQ ID NO:9 and SEQ ID NO:10. The resulting promoter DNA was integrated into the plasmid sequence using an In-Fusion HD Cloning Kit (Takara). The cellulase sequence was removed from the resulting plasmid sequence by PCR using the primer set of SEQ ID NO:11 and SEQ ID NO:12. Using the resulting plasmid as a template, the plasmid sequence was amplified by PCR using the primer set of SEQ ID NO:11 and SEQ ID NO:13. Using the genome of strain 168 as a template, secretion signal DNA was amplified by PCR using a primer set of SEQ ID NO: 14 and SEQ ID NO: 15. The obtained secretion signal DNA was integrated into a plasmid sequence using an In-Fusion HD Cloning Kit (Takara) to construct a PPIase expression plasmid.

[0058] 1-2. Design of PPIase Expression Plasmid Using the PPIase expression plasmid constructed in 1-1 as a template, the plasmid sequence was amplified by PCR using the primer set of SEQ ID NO: 11 and SEQ ID NO: 13 and PrimeSTAR Max DNA polymerase (TaKaRa). Using the genome of strain 168 as a template, DNA encoding PrsA shown in SEQ ID NO: 18 was amplified by PCR using the primer set of SEQ ID NO: 16 and SEQ ID NO: 17 (SEQ ID NO: 19). The resulting DNA encoding PrsA was integrated into the plasmid sequence using In-Fusion HD Cloning Kit (Takara) to obtain a PrsA expression plasmid. Using the PPIase expression plasmid constructed in 1-1 as a template, the plasmid sequence was amplified by PCR using the primer set of SEQ ID NO: 13 and SEQ ID NO: 20 and PrimeSTAR Max DNA polymerase (TaKaRa). Using the genome of Escherichia coli DH5α as a template, DNAs encoding SurA (SEQ ID NO:29), PpiA (SEQ ID NO:30), Tig (SEQ ID NO:31), and FkpA (SEQ ID NO:1) were amplified by PCR using primer sets of SEQ ID NOs:21 and 22, 23 and 24, 25 and 26, and 27 and 28 (SEQ ID NOs:32 to 35). The resulting DNAs encoding PPIase were integrated into plasmid sequences using an In-Fusion HD Cloning Kit (Takara) to obtain plasmids for expressing SurA, PpiA, Tig, and FkpA, respectively.The nucleotide sequences shown in SEQ ID NOs: 40 to 48, which encode the amino acid sequences of FKBP_l (SEQ ID NO: 2), FKBP_K (SEQ ID NO: 3), FKBP_E (SEQ ID NO: 4), FKBP_P (SEQ ID NO: 5), FKBP2 (SEQ ID NO: 36), FK153 (SEQ ID NO: 37), MJFKS (SEQ ID NO: 6), FKBP52 (SEQ ID NO: 38), and CyP40 (SEQ ID NO: 39), were synthesized by GenPlus cloning from Genescript Inc., by incorporating them between the regions shown in SEQ ID NOs: 49 and 50 of the PPIase expression plasmid constructed in 1-1, to obtain plasmids for expression of FKBP_l, FKBP_K, FKBP_E, FKBP_P, FKBP2, FK153, MJFKS, FKBP52, and CyP40, respectively.

[0059] 1-3. Construction of Fab Expression Strains Using the genome of strain 168 as a template, a DNA fragment (A) containing the ybdO gene region and a DNA fragment (B) containing the ybxG gene region were amplified by PCR using primer sets of SEQ ID NOs: 51 and 52, and SEQ ID NOs: 53 and 54. Using plasmid pC194 DNA as a template, a chloramphenicol resistance gene region (C) was amplified by PCR using a primer set of SEQ ID NOs: 55 and 56. Fab_c (Certolizumab Fab) consisting of the heavy and light chains shown in SEQ ID NOs: 57 and 58 was synthesized by cloning the nucleotide sequence shown in SEQ ID NO: 59, which can be expressed in an operon, between the regions shown in SEQ ID NOs: 60 and 50 of the PPIase expression plasmid constructed in 1-1 using Genescript's GenPlus cloning software, to obtain a Fab_c expression plasmid. Using the Fab_c expression plasmid as a template, a DNA fragment (D) containing the Fab_c expression region was amplified by PCR using a primer set of SEQ ID NO: 61 and SEQ ID NO: 62. The four fragments (A), (B), (C), and (D) were mixed as a template, and SOE-PCR was performed using a primer set of SEQ ID NO: 51 and SEQ ID NO: 54 to ligate the four fragments and obtain a DNA fragment for genome integration of the Fab_c expression region. The resulting DNA fragment for genome integration of the Fab_c expression region was transformed into the Bacillus subtilis Dpr9ΔsigF strain by the competent cell method. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants. The strain obtained in this manner was designated the Fab_c-producing strain.

[0060] 1-4. Introduction of PPIase Expression Plasmid into Bacillus subtilis The PPIase expression plasmid constructed in 1-2 was introduced into the Fab_c production strain using the protoplast method described below. Glycerol-stocked B. subtilis was inoculated into 1 mL of LB liquid medium and cultured overnight with shaking at 30°C and 210 rpm. The next day, 10 μL of this culture was inoculated into a fresh 1 mL of LB liquid medium and cultured with shaking at 37°C and 210 rpm for approximately 2 hours. This culture was collected in a 1.5 mL tube and centrifuged at 1,2000 rpm for 5 minutes. The supernatant was removed, and the pellet was suspended in 500 μL of SMMP containing 4 mg / mL of Lysozyme (Sigma) and incubated at 37°C for 1 hour. The mixture was then centrifuged at 3,500 rpm for 10 minutes, and the supernatant was removed. The resulting pellet was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with the various plasmids, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to the mixture, and the mixture was mixed by inversion. After shaking at 30°C and 210 rpm for 1 hour, the entire mixture was spread on a DM3 agar plate and incubated at 30°C for 2-3 days.

[0061] 1-5. Fab Production The recombinant Bacillus subtilis prepared in 1-4 was inoculated into 500 μL of LB medium containing 50 ppm tetracycline and cultured overnight at 30°C in a 96-well plate to prepare a preculture solution. The preculture solution was inoculated at 1% into 800 μL of 2×L-mal medium containing 15 ppm tetracycline and cultured in a 96-well plate at 30°C for 72 hours. At the end of the culture, the plate was centrifuged at 4°C, 3,000 rpm, and 20 minutes, and the supernatant was collected.

[0062] 1-6. Quantification of Fab Production by ELISA 100 μL of 1 μg / mL TNF-α (Wako) was added to each well of an F96 Cert. Maxisorp Nunc-Immuno™ Plate (Thermo Fisher Scientific), sealed, and allowed to stand overnight at 4°C. After removing any TNF-α that had not adsorbed to the wells, 200 μL of PBST (PBS containing 0.1% (v / v) Tween 20) was added and immediately removed. This washing procedure was performed three times. PBST containing 2% skim milk was added, and the plate was incubated at room temperature for 1 hour. After removing the PBST containing skim milk, 200 μL of PBST was added and immediately removed. This washing procedure was performed three times. The culture supernatant collected in 1-5 was diluted 50-fold with PBST and further diluted 10-fold with PBST containing 2% skim milk. A similar dilution procedure was performed on the dilution series of Certolizumab Recombinant Human Monoclonal Antibody (Invitrogen). 100 μL of the diluted solution was added to each well containing immobilized TNF-α and incubated at room temperature for 1 hour. After removing the diluted solution, 200 μL of PBST was added and immediately removed. This washing procedure was performed three times. Goat anti-human IgG (H+L) secondary antibody, HRP (Invitrogen) was used as the detection antibody. The detection antibody was diluted 1 / 5,000 with PBST. 100 μL of detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST was added and immediately removed. This washing procedure was repeated three times. The chromogenic substrate was prepared by dissolving an OPD tablet (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of chromogenic substrate was added to each well and incubated for 10 minutes in the dark. After adding 100 μL of 0.5 mol / L sulfuric acid, the absorbance at 490 nm was measured using a microplate reader.The amount of Fab_c produced was calculated based on a dilution series of Certolizumab Recombinant Human Monoclonal Antibody (Invitrogen), and the relative values ​​compared to the Fab_c-producing strain into which pHY300PLK had been introduced were shown (Figure 1). As a result, it was confirmed that the amount of Fab_c produced was significantly improved in the Fab_c-producing strain harboring the plasmids for expression of FkpA, FKBP_1, FKBP_K, FKBP_E, FKBP_P, and MJFKS. The amount of Fab_c produced was reduced in the Fab_c-producing strain harboring the plasmid for expression of PrsA, an endogenous factor of Bacillus subtilis. Fab_c has a prolyl bond isomerization site, specifically, a cis-type prolyl bond.

[0063] 1-7. Confirmation of Fab production by Western blotting The culture supernatant obtained in 1-5 was mixed with an equal volume of Laemmli Sample Buffer (BIO-RAD), and then heat-treated at 99°C for 5 minutes to prepare a sample. ＴＭ Mini-PROTEAN TGX ＴＭPrecast Gel (BIO-RAD) was used. Five microliters of sample was applied to each well and electrophoresed at 210 V for 5 minutes. Precision Plus Blue standard (BIO-RAD) was used as a molecular weight marker, and Certolizumab Recombinant Human Monoclonal Antibody (Invitrogen) was also run simultaneously as a standard. Proteins were transferred from the SDS-PAGE gel to a PVDF membrane using Trans-Blot Turbo Mini PVDF Transfer Packs (BIO-RAD) and a Trans-Blot Turbo System (BIO-RAD). The antibody used was Goat anti-Human IgG (H+L) Secondary Antibody, HRP (Invitrogen), and the iBind Western System (Invitrogen) was used for the antibody reaction. The target protein was detected using 1-Step Ultra TMB-Blotting Solution (Thermo Scientific) (Figure 2). As with the ELISA quantification shown in 1-6, it was confirmed that the productivity of the Fab_c production strain harboring the FkpA expression plasmid was improved compared to the Fab_c production strain into which pHY300PLK had been introduced. It was also confirmed that the productivity of the Fab_c production strain harboring the PrsA expression plasmid was reduced compared to the Fab_c production strain into which pHY300PLK had been introduced.

[0064] Example 2 Verification of domains necessary for high production 2-1. Design of plasmids for expressing each domain Using the FkpA expression plasmid constructed in 1-2 as a template, the C-terminal region or N-terminal region of FkpA was removed by PCR using primer sets of SEQ ID NOs: 63 and 64, and SEQ ID NOs: 65 and 66, and PrimeSTAR Max DNA polymerase (TaKaRa), to obtain an FkpA_N expression plasmid expressing the N-terminal region (SEQ ID NO: 67) of FkpA and an FkpA_C expression plasmid expressing the C-terminal region (SEQ ID NO: 68), respectively.

[0065] 2-2. Introduction of PPIase Expression Plasmids into Bacillus subtilis The plasmids for expressing each domain constructed in 2-1 were introduced into the Fab_c production strain by the protoplast method described in 1-4.

[0066] 2-3. Fab Production The recombinant Bacillus subtilis prepared in 2-2 was inoculated into 500 μL of LB medium containing 50 ppm tetracycline and cultured overnight at 30°C in a 96-well plate to prepare a preculture solution. The preculture solution was inoculated at 1% into 800 μL of 2×L-mal medium containing 15 ppm tetracycline and cultured in a 96-well plate at 30°C for 72 hours. At the end of the culture, the plate was centrifuged at 4°C, 3000 rpm, and 20 minutes, and the supernatant was collected.

[0067] 2-4. Quantification of Fab Production Amount by ELISA The production amount of Fab_c was quantified by the ELISA method described in 1-6. The production amount of Fab_c was calculated based on the dilution series concentration of Certolizumab Recombinant Human Monoclonal Antibody (Invitrogen), and the relative value to the Fab_c-producing strain into which pHY300PLK had been introduced is shown (Figure 3). As a result, it was confirmed that in order to obtain a significant productivity improvement effect in the Bacillus subtilis production system, it is necessary to express both the N-terminal and C-terminal regions of FkpA.

[0068] Example 3: Verification of the effect of PPIase localization on protein production enhancement 3-1. Construction of Fab expression plasmid Using recombinant plasmid pHY-S237 (JP 2014-158430 A), which was constructed based on pHY300PLK, as a template, the plasmid sequence was amplified by PCR using a primer set of SEQ ID NO: 7 and SEQ ID NO: 8 and PrimeSTAR Max DNA polymerase (TaKaRa). Using the genome of strain 168 as a template, promoter DNA derived from the spoVG gene was amplified by PCR using a primer set of SEQ ID NO: 9 and SEQ ID NO: 10. The resulting promoter DNA was integrated into the plasmid sequence using an In-Fusion HD Cloning Kit (Takara). Using the resulting plasmid as a template, the plasmid sequence was amplified by PCR using a primer set of SEQ ID NO: 11 and SEQ ID NO: 13. The base sequence shown in SEQ ID NO: 71, which enables Fab_l (Fab against lysozyme) consisting of the heavy chain and light chain shown in SEQ ID NO: 69 and SEQ ID NO: 70 to be expressed in an operon, was artificially synthesized at Thermo Fisher Scientific, and incorporated into a plasmid sequence using an In-Fusion HD Cloning Kit (Takara), thereby constructing a plasmid for expressing Fab_l.

[0069] 3-2. Construction of PPIase-Expressing Strains Using the genome of strain 168 as a template, a DNA fragment containing the bglP gene region (A), a DNA fragment containing the yxxE gene region (B), and promoter DNA derived from the spoVG gene (C) were amplified by PCR using primer sets of SEQ ID NOs: 72 and 73, 74 and 75, and 76 and 77. Using plasmid pC194 DNA as a template, a chloramphenicol resistance gene region (D) was amplified by PCR using primer sets of SEQ ID NOs: 55 and 56. Using the genome of E. coli DH5α as a template, a DNA encoding FkpA linked to a secretion signal or a signal for anchoring a protein on a membrane was amplified by PCR using primer sets of SEQ ID NOs: 78 and 79, and SEQ ID NOs: 79 and 80 (E) (E'). The five fragments (A), (B), (C), (D), and (E) or (A), (B), (C), (D), and (E') above were mixed as a template, and SOE-PCR was performed using the primer set of SEQ ID NO: 72 and SEQ ID NO: 75 to ligate the five fragments and obtain a DNA fragment for genome integration that expresses FkpA via secretion or membrane anchoring. The resulting DNA fragment for genome integration was used to transform the Bacillus subtilis Dpr9ΔsigF strain by the competent cell method. After transformation, colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants. The resulting strain capable of secreting and expressing FkpA was designated the FkpA_p strain, and the strain capable of expressing FkpA via membrane anchoring was designated the FkpA_m strain.

[0070] 3-3. Introduction of Fab Expression Plasmid into Bacillus subtilis The Fab_1 expression plasmid constructed in 3-1 was introduced into the FkpA_p and FkpA_m strains by the protoplast method described in 1-4.

[0071] 3-4. Fab Production The recombinant Bacillus subtilis prepared in 3-3 was inoculated into 500 μL of LB medium containing 50 ppm tetracycline and cultured overnight at 30°C in a 96-well plate to prepare a preculture solution. The preculture solution was inoculated at 1% into 800 μL of 2×L-mal medium containing 15 ppm tetracycline and cultured in a 96-well plate at 30°C for 72 hours. At the end of the culture, the plate was centrifuged at 4°C, 3,000 rpm, and 20 minutes, and the supernatant was collected.

[0072] 3-5. Quantification of Fab Production by ELISA 100 μL of 100 μg / mL lysozyme from Egg White (Wako) was added to each well of an F96 Cert. Maxisorp Nunc-Immuno™ Plate (Thermo Fisher Scientific), sealed, and allowed to stand overnight at 4°C. After removing any lysozyme that had not adsorbed to the wells, 200 μL of PBST (PBS containing 0.1% (v / v) Tween 20) was added and immediately removed. This washing procedure was repeated three times. PBST containing 2% skim milk was added, and the plate was incubated at room temperature for 1 hour. After removing the PBST containing skim milk, 200 μL of PBST was added and immediately removed. This washing procedure was performed three times. The culture supernatant collected in 3-4 was diluted 10-fold with PBST containing 2% skim milk. 100 μL of the diluted solution was added to each well containing lysozyme and incubated at room temperature for 1 hour. After removing the diluted solution, 200 μL of PBST was added and immediately removed. This washing procedure was performed three times. Mouse IgG Fab Antibody:HRP (Biovallley) was used as the detection antibody. The detection antibody was diluted 1 / 5,000 with PBST. 100 μL of the detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST was added and immediately removed. This washing procedure was performed three times. The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the chromogenic substrate was added to each well and incubated for 20 minutes in the dark. After adding 100 μL of 0.5 mol / L sulfuric acid, the absorbance at 490 nm was measured using a Microplate Reader. The absorbance values ​​obtained are shown relative to those of a strain in which the Fab_l expression plasmid was introduced into Dpr9ΔsigF (Figure 4).As a result, it was revealed that secretion of FkpA contributes to higher production of the target protein than membrane-anchored expression, even though Bacillus subtilis is known to fold proteins near the cell membrane. Fab_1 has a prolyl bond isomerization site, specifically, a cis-type prolyl bond.

[0073] Example 4 Increased Protein Production by PPIase Expression 4-1. Construction of Plasmid for RNase T1 Expression The nucleotide sequence shown in SEQ ID NO: 101, which encodes the amino acid sequence of RNase T1 (SEQ ID NO: 100), was synthesized by GenPlus cloning from Genescript Inc. so as to be inserted between the regions shown in SEQ ID NO: 49 and SEQ ID NO: 50 of the PPIase expression plasmid constructed in 1-1, thereby obtaining a plasmid for RNase T1 expression.

[0074] 4-2. Introduction of RNase T1 expression plasmid into PPlase expression strain Introduction of the RNase T1 expression plasmid into the Bacillus subtilis Dpr9ΔsigF strain and the FkpA_p strain constructed in 3-2 was carried out by the protoplast method described in 1-4.

[0075] 4-3. RNase T1 Production The recombinant Bacillus subtilis prepared in 4-2 was inoculated into 500 μL of LB medium containing 50 ppm tetracycline and cultured overnight at 30°C in a 96-well plate to prepare a preculture solution. The preculture solution was inoculated at 1% into 800 μL of 2xL-mal medium containing 15 ppm tetracycline and cultured in a 96-well plate at 30°C for 72 hours. At the end of the culture, the plate was centrifuged at 4°C, 3000 rpm, and 20 minutes, and the supernatant was collected.

[0076] 4-4. Quantification of RNase T1 production by SDS-PAGE Equal volumes of the culture supernatant collected in 4-3 and Laemmli Sample Buffer (BIO-RAD) were mixed and then heated at 99°C for 5 minutes to prepare a sample. Mini-PROTEIN TGX Stain-Free (BIO-RAD) was used for the gel. 5 μL of sample was applied to each well and electrophoresed at 210 V for 25 minutes. Precision Plus protein unstained standard (BIO-RAD) was used as a molecular weight marker. Protein bands were detected using a ChemiDoc MP Imaging System. The detected protein was quantified using a calibration curve prepared based on a lysozyme standard (Sigma-Aldrich) (Table 1). The results confirmed that expression of PPIase significantly increased the production of RNase T1. RNase T1 has a prolyl bond isomerization site, specifically, a cis-type prolyl bond.

[0077]

[0078] Reference Example 1 Identification of PPIase Sequences Belonging to the BCFK Clade Using HMM Profiles Phylogenetic Analysis of FKBP Phylogenetic analysis was performed using a group of sequences in which the FkpA sequence shown in SEQ ID NO: 81 was added to the FKBP sequences registered as seed sequences for the PF00254 family in Pfam shown in Table 2. After constructing a multiple alignment using the default parameters of MAFFT v7.471, conserved regions were extracted using trimAl v1.4.rev15, and an evolutionary model was selected using ModelTest-NG v0.1.7. IQ-TREE multicore version 2.0.3 was used for the phylogenetic analysis, and LG+G4+F was specified as the evolutionary model. A phylogenetic tree visualized using Figtree (http: / / tree.bio.ed.ac.uk / software / FigTree / ) is shown in Figure 5. Table 3 shows the Uniprot numbers of the FKBP sequences belonging to the BCFK clade in Figure 5 .

[0079]

[0080]

[0081] (2) Identification of FKBP sequences belonging to the BCFK clade. A file named BCFK.fasta was prepared, which lists the FKBP sequences belonging to the BCFK clade shown in Table 3 in Fasta format. A multiple alignment was constructed using MAFFT v7.471 with the command mafft --auto BCFK_mafft.fasta>BCFK.fasta. Next, using HMMER version 3.3.2, the command hmmbuild --pnone --wnone BCFK_mafft.hmm BCFK_mafft.fasta was executed to create the HMM profile shown in Figure 6, and the command hmmpress BCFK_mafft.hmm was executed to binarize the profile. A file named No_BCFK.Fasta was prepared, which lists the FKBP sequences in Table 2 that do not belong to the BCFK clade in Fasta format. By executing the command hmmscan --tblout BCFK.tblout BCFK_mafft.hmm No_BCFK.Fasta, a homology search was performed for FKBP sequences not included in the BCFK clade against the HMM profile defining the BCFK clade. As a result, it was confirmed that all sequences not included in the BCFK clade had an HMM score of 112.3 or less. Next, a file named Express_PPIase.Fasta was prepared, which compiled the PPIase sequences represented by SEQ ID NOs: 18, 81 to 93 (PrsA, FkpA, FKBP_1, FKBP_K, FKBP_E, FKBP_P, MJFKS, SurA, PpiA, Tig, FKBP2, FK153, FKBP52, CyP40) in Fasta format. By executing the command hmmscan --tblout BCFK.tblout BCFK_mafft.hmm Express_PPIase.Fasta, a homology search of the PPIase sequence described in Example 1 was performed against the HMM profile defining the BCFK clade. Sequences with no homology were given an HMM score of 0. The results are shown in Table 4. FkpA, FKBP_1, FKBP_K, FKBP_E, and FKBP_P, which showed a high productivity improvement effect in 1-6 of Example 1, have HMM scores of 150 or more, and can be said to be FKBP sequences belonging to the BCFK clade.In this way, it is possible to confirm whether any PPIase sequence is a PPIase sequence belonging to the BCFK clade.

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Claims

1. A method for producing a target protein, comprising culturing a Gram-positive bacterium containing a gene encoding a heterologous FK506 binding protein (FKBP) and a gene encoding a target protein, or co-culturing a Gram-positive bacterium containing a gene encoding a heterologous FKBP and a Gram-positive bacterium containing a gene encoding a target protein.

2. The method according to claim 1, wherein the heterologous FKBP is a polypeptide belonging to the BCFK clade and having prolyl bond isomerization activity, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 6 and having prolyl bond isomerization activity.

3. The method according to claim 1, wherein the heterologous FKBP is a polypeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence having at least 80% identity with an amino acid sequence shown in any one of SEQ ID NOs: 1 to 6 and having prolyl bond isomerization activity.

4. The method according to any one of claims 1 to 3, wherein the gene encoding the heterologous FKBP is operably linked to a polynucleotide encoding a secretory signal peptide.

5. The method according to any one of claims 1 to 4, wherein the Gram-positive bacterium is Bacillus subtilis or a mutant thereof.

6. The method according to any one of claims 1 to 4, wherein the Gram-positive bacterium is Bacillus subtilis in which at least one extracellular protease gene has been deleted or inactivated.

7. The method according to any one of claims 1 to 6, wherein the target protein has at least one prolyl bond isomerization site.

8. The method according to any one of claims 1 to 7, wherein the target protein is an antibody-related molecule.

9. The method according to any one of claims 1 to 7, wherein the target protein is a Fab.

10. A Gram-positive bacterium containing a gene encoding a heterologous FKBP, or a gene encoding a heterologous FKBP and a gene encoding a protein of interest.

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