Modified signal peptide

Abstract

To provide a modified signal peptide, and secretion and production of a target protein using the same.SOLUTION: A nucleic acid construct includes a polynucleotide encoding a target protein and a polynucleotide encoding a modified signal peptide linked upstream of the polynucleotide, the modified signal peptide having amino acid residues that are first to sixth from a C-terminal of X1X2X3X4X5X6, where X1 is G, P, or A, X2 is G, S, or A, X3 is P, S, or A, provided that X1X2X3 is not GGP, X4 is A, X5 is S, H, or F, and X6 is A, the nucleic acid construct located at the C-terminal of the modified signal peptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to modified signal peptides. [Background technology]

[0002] Bacillus bacteria such as Bacillus subtilis have the function of secreting and producing proteins such as proteases, and are therefore suitable as hosts for the microbial production of target substances. Secretory proteins in Bacillus bacteria are expressed as preproproteins having a signal peptide, and are secreted outside the cells by the action of the signal peptide. The preproprotein is secreted outside the cells after the signal peptide is cleaved by a signal peptidase, and finally becomes a mature protein. A technique is used in which a signal peptide derived from Bacillus bacteria is linked to a target protein and secreted outside the cells. For example, the signal peptide of cellulase derived from Bacillus sp. KSM-S237 strain (Patent Document 1) is a suitable signal peptide for efficient secretory production of a target protein.

[0003] A signal peptide generally consists of an N-terminal region with a positive charge, a hydrophobic membrane-spanning region (H region) that forms an α-helix, and a C-terminal region with a sheet-like structure. The peptide in the C-terminal region is mainly cleaved by a signal peptidase. It has been reported that modification of a signal peptide can affect protein productivity (e.g., Non-Patent Document 1).

[0004] Low molecular weight antibodies have advantages such as production efficiency, ease of drug design, and tissue permeability, and therefore have been attracting attention as materials for antibody drugs and reagents. Representative low molecular weight antibodies include Fab, single chain antibody (scFv), diabody, and variable domain of heavy chain of heavy chain antibody (VHH). Conventionally, Escherichia coli has been mainly used for the microbiological production of antibodies. On the other hand, Non-Patent Document 2 describes that scFv, Fab, and VHH were produced by a Brevibacillus expression system. Non-Patent Document 3 reports that VHH was produced using Aspergillus awamori. Non-Patent Document 4 describes that a Bacillus subtilis strain co-expressing intracellular and extracellular molecular chaperones and inactivating the cell wall-bound protease WprA secreted and produced scFv derived from a fibrin-specific monoclonal antibody. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2000-210081 A [Non-patent literature]

[0006] [Non-Patent Document 1] Protein Sci, 2012, 21(1):13-25 [Non-Patent Document 2] Bioengineering, 2017, 95(11):649-651 [Non-Patent Document 3] Appl Microbiol Biotechnol, 2005, 66(4):384-392 [Non-Patent Document 3] Appl Environ Microbiol, 2002, p.3261-3269 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to a modified signal peptide and the secretory production of a target protein using the same. [Means for solving the problem]

[0008] In one aspect, the present invention relates to a method for producing a gene encoding a target protein, comprising the steps of: (a) providing a gene encoding a target protein; and (b) providing a gene encoding a target protein; The modified signal peptide has 1 to 6 amino acid residues from the C-terminus of X1X2X3X4X5X6, where X1 is G, P or A, X2 is G, S or A, X3 is P, S or A, with the proviso that X1X2X3 is not GGP, X4 is A, X5 is S, H or F, and X6 is A, and is located at the C-terminus of the modified signal peptide. A nucleic acid construct for expressing and secreting a target protein outside a cell is provided. In another aspect, the present invention provides an expression vector comprising the nucleic acid construct. In another aspect, the present invention provides a recombinant Bacillus comprising the nucleic acid construct. In a further aspect, the present invention relates to a method for producing a recombinant Bacillus sp. comprising culturing the recombinant Bacillus sp. Recovering the target protein secreted outside the cell. The present invention provides a method for producing a target protein, comprising the steps of: In a further aspect, the present invention relates to a polypeptide encoding an antibody-related molecule linked to the modified signal peptide of the present invention on the C-terminal side thereof, The modified signal peptide has 1 to 6 amino acid residues from the C-terminus of X1X2X3X4X5X6, where X1 is G, P or A, X2 is G, S or A, and X3 is P, S or A, with the proviso that X1X2X3 is not GGP, X4 is A, X5 is S, H or F, and X6 is A, which are located at the C-terminus of the modified signal peptide; and the amino acid sequence from the 7th amino acid from the C-terminus to the N-terminus of the modified signal peptide consists of any one of the amino acid sequences of SEQ ID NOs: 7 to 12, or an amino acid sequence at least 90% identical thereto; Preproproteins of antibody-related molecules are provided. Effect of the Invention

[0009] The modified signal peptide provided by the present invention is useful for the secretion and production of a target protein. The modified signal peptide enables a target protein such as an antibody-related molecule to be efficiently secreted outside the cell and produced in high yield. Furthermore, the antibody-related molecule secreted using the modified signal peptide has almost no unnecessary residues, such as signal peptide residues, remaining at the N-terminus, making it highly valuable as a raw material for various medicines and reagents. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As used herein, the term "antibody-related molecule" refers to a protein containing an immunoglobulin or a molecular species consisting of a single domain selected from the domains constituting an immunoglobulin or a combination of two or more domains. The domains constituting an immunoglobulin include VH, CH1, CH2, and CH3, which are domains of an immunoglobulin heavy chain, and VL and CL, which are domains of an immunoglobulin light chain. 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 antigen recognition domain of an immunoglobulin and can specifically bind to a target such as a protein. Examples of antibody-related molecules include immunoglobulins such as IgG, as well as small molecule antibodies such as Fab, F(ab')2, single chain antibodies (scFv), diabodies, and variable domains of heavy chains of heavy chain antibodies (VHH).

[0011] As used herein, "VHH" refers to the variable domain of a heavy chain 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 antibody). The VHH in this specification is derived from, for example, camelids or sharks, preferably from camelids. VHH is a molecule capable of recognizing an antigen with a single domain, and is the smallest unit of antibody molecules found to date. The VHH produced in 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 in 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 by 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.

[0012] In this specification, the camelid family includes Bactrian camels, dromedaries, llamas, alpacas, vicunas, guanacos, etc., and preferably includes alpacas.

[0013] In the present specification, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using the search homology program of the genetic information processing software Genetyx-Win with the unit size to compare (ktup) set to 2.

[0014] As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more.

[0015] Unless otherwise defined herein, "one or several" used in relation to deletion, substitution, addition, or insertion of amino acid residues in an amino acid sequence may preferably mean 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. Furthermore, unless otherwise defined herein, "one or several" used in relation to deletion, substitution, addition, or insertion of nucleotides in a nucleotide sequence may preferably mean 1 to 15, more preferably 1 to 9, and even more preferably 1 to 6. As used herein, "addition" of an amino acid residue or nucleotide includes addition of an amino acid residue or nucleotide to one end and both ends of a sequence.

[0016] In the present specification, the "corresponding position" or "corresponding region" on 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) so as to provide maximum 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. Clustal W can be used, for example, on the website of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) or the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics. A position of a target sequence that is aligned to any position of a reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" that position. In addition, a region flanked by corresponding positions or a region consisting of a corresponding motif is considered to be a corresponding region.

[0017] Those skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above to optimize it. Such an optimal alignment is preferably determined taking into consideration the identity or similarity of the amino acid sequences, the frequency of gaps to be inserted, and the like. Here, the identity or similarity of the amino acid sequences refers to the ratio (%) of the number of positions at which identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned to the total number of amino acid residues. The term "similar amino acid residues" refers to amino acid residues that have similar properties in terms of polarity and charge among the 20 types of amino acids that constitute proteins, and that cause so-called conservative substitutions. Such groups of similar amino acid residues are well known to those skilled in the art, and examples thereof include, but are not limited to, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; leucine and isoleucine.

[0018] As used herein, the term "amino acid residue" refers to the 20 types of amino acid residues constituting 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).

[0019] In this specification, the term "expression cassette" refers to a nucleic acid construct for controlling the expression of a gene of interest contained therein. Preferably, the expression cassette of the present invention is a DNA construct. Usually, an expression cassette contains a coding region of a gene of interest to be expressed and a control region for controlling the expression, such as a promoter. Most of the control region is located upstream of the coding region of the gene of interest and is operably linked thereto. For example, when the gene of interest is a polynucleotide encoding a preproprotein containing a signal peptide, the promoter is located upstream of the region encoding the signal peptide, operably linked to the polynucleotide encoding the preproprotein, and controls the expression of the preproprotein. The expression cassette may also contain the 3' untranslated region and 5' untranslated region of the gene of interest. Preferably, the expression cassette has a restriction enzyme recognition site at its end to enable the insertion and / or excision of the expression cassette into a vector or genomic DNA. The expression cassette can be used for constructing an expression vector or introducing a foreign gene into genomic DNA.

[0020] As used herein, a "control region" refers to a region that has the function of controlling the expression of a gene (e.g., a region that codes for a protein or peptide) located downstream of the control region. More specifically, a "control region" can be defined as a region that exists upstream of a coding region of a gene and has the function of controlling the transcription of the coding region by interacting with RNA polymerase. The control region includes a transcription initiation control region and / or a translation initiation control region, or a region from the transcription initiation control region to the translation initiation control region. The transcription initiation control region is a region that includes a promoter and a transcription start point, and the translation initiation control region is a region that corresponds to the Shine-Dalgarno (SD) sequence that forms a ribosome binding site together with an initiation codon (Proc Natl Acad Sci USA, 1974, 71:1342-1346).

[0021] As used herein, the term "operably linked" between a regulatory region and a polynucleotide of a gene (e.g., a polynucleotide encoding a protein) refers to the linkage between the gene and the regulatory region such that the gene can be expressed under the control of the regulatory region. Procedures for "operably linked" between a gene and a regulatory region are well known to those skilled in the art.

[0022] As used herein, "upstream" and "downstream" in relation to a gene or its nucleic acid sequence refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter in the DNA sense strand, and "upstream" of a gene means the 5' region of the gene in the DNA sense strand.

[0023] A "signal peptide" has the function of secreting a target protein operably linked thereto outside the cell. Such a target protein is first expressed as a preproprotein having a signal peptide, and is secreted outside the cell via the action of the signal peptide. The preproprotein is secreted outside the cell after the signal peptide is cleaved by a signal peptidase, and finally becomes a mature protein. A signal peptide generally consists of an N-terminal region having a positive charge, a hydrophobic membrane-spanning region (H region) that forms an α-helix, and a C-terminal region having a sheet-like structure (Protein Science, 2012, 21:13-25). The peptide in the C-terminal region is mainly cleaved by the signal peptidase.

[0024] As used herein, "operably linked" between the amino acid sequence of a target protein and a signal peptide means that the amino acid sequence of the target protein is linked to the signal peptide so that it can be secreted outside the cell under the control of the signal peptide. The procedure for "operably linked" is well known to those skilled in the art, and the amino acid sequence of the target protein is usually linked to the C-terminus of the signal peptide. Another sequence (e.g., an amino acid sequence that is not secreted outside the cell by processing) may be included between the amino acid sequence of the target protein that is operably linked to the signal peptide.

[0025] As used herein, the term "native" when referring to a function, property, or trait of a cell is used to indicate that the function, property, or trait is inherently present in the cell. In contrast, the term "exogenous" is used to indicate a function, property, or trait that is not inherently present in the cell, but is introduced from the outside. For example, an "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from the outside. An exogenous gene or polynucleotide may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).

[0026] As used herein, the phrase "having the correct N-terminus" of a secreted protein means that at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% (all w / w%) of the protein secreted outside the cell via a preproprotein containing a signal peptide does not have amino acid residues at the N-terminus that the protein does not originally have (e.g., remaining signal peptide residues).

[0027] In this specification, the term "bacteria of the genus Bacillus" refers to bacteria of the genus Bacillus in the family Bacillaceae. Examples of bacteria of the genus Bacillus include Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus liqueniformis, and mutant strains thereof. Note that bacteria of the genera Paenibacillus and Brevibacillus belonging to the family Paenibacillaceae are not included in the genus Bacillus in this specification.

[0028] The names of the genes of Bacillus subtilis 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 gene numbers of Bacillus subtilis described herein refer to the gene numbers registered in BSORF DB.

[0029] Unless otherwise specified, the gene or protein names of the antibody-related molecules described herein are in accordance with the registration information in the Protein Data Bank (PDB) ([www.rcsb.org / ]).

[0030] The present invention provides a modified signal peptide. The modified signal peptide of the present invention can be produced by modifying the C-terminal region of a parent signal peptide. Examples of the parent signal peptide include signal peptides that function in Bacillus bacteria, and preferred examples include signal peptides derived from Bacillus bacteria. Preferred examples of signal peptides derived from Bacillus bacteria include the signal peptides of Bacillus subtilis YoaW, lipA, pelB, and YncM (SEQ ID NO: 1 to 4), the signal peptide of cellulase derived from Bacillus sp. KSM-S237 strain (SEQ ID NO: 5), and the signal peptide derived from amylase of Bacillus amyloliquefaciens (SEQ ID NO: 6). Another preferred example of the parent signal peptide is a signal peptide consisting of an amino acid sequence at least 90% identical to any of SEQ ID NO: 1 to 6. Among these, a signal peptide consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or an amino acid sequence at least 90% identical thereto, is preferred as the parent signal peptide.

[0031] The C-terminal region of a signal peptide can be predicted using three-dimensional structure prediction by CRNPRED ([pdbj.org / crnpred]). A region present at the C-terminus of a signal peptide that is predicted not to have an α-helical structure can be predicted as the C-terminal region. For example, the C-terminal region of the parent signal peptide corresponds to residues 17 to 24 in SEQ ID NO: 1, residues 27 to 32 in SEQ ID NO: 2, residues 19 to 24 in SEQ ID NO: 3, residues 35 to 42 in SEQ ID NO: 4, residues 24 to 29 in SEQ ID NO: 5, and residues 25 to 31 in SEQ ID NO: 6.

[0032] Preferably, the C-terminal region of the modified signal peptide of the present invention is composed of a sequence of 6 amino acid residues including modified amino acid residues. More specifically, the 1st to 6th amino acid residues from the C-terminus of the modified signal peptide are X1X2X3X4X5X6, where X6 is the residue located at the C-terminus of the modified signal peptide and X1 is the residue located at the 6th position from the C-terminus. Residues X1 to X6 are as follows: X1: preferably G, P or A; X2: preferably G, S or A, more preferably G; X3: preferably P, S or A, more preferably P or S; X4: preferably A; X5: preferably S, H or F, more preferably S or H; X6:Preferably A. Preferably, X1X2X3 is not GGP. Preferred examples of the sequence of X1X2X3X4X5X6 include any of the amino acid sequences of SEQ ID NOs: 13 to 30, more preferred examples include any of the amino acid sequences of SEQ ID NOs: 14 to 30, and even more preferred examples include any of the amino acid sequences of SEQ ID NOs: 15, 25 to 28.

[0033] The region other than the C-terminal region of the modified signal peptide of the present invention may consist of the same amino acid sequence as the parent signal peptide, or alternatively, the portion of the modified signal peptide other than the C-terminal region may consist of an amino acid sequence in which one or several amino acid residues have been deleted, substituted, added or inserted relative to the amino acid sequence of the parent signal peptide, so long as the function as a signal peptide is not lost. Preferably, the modified signal peptide of the present invention has a region from the C-terminus to the 7th residue to the N-terminus that consists of an amino acid sequence of a region other than the C-terminal region of a Bacillus signal peptide, or an amino acid sequence at least 90% identical thereto. More preferably, the modified signal peptide of the present invention has a region from the C-terminus to the 7th residue to the N-terminus that consists of an amino acid sequence of any one of SEQ ID NOs: 7 to 12, or an amino acid sequence at least 90% identical thereto. Even more preferably, the modified signal peptide of the present invention has a region from the C-terminus to the 7th residue to the N-terminus that consists of an amino acid sequence of SEQ ID NO: 10 or 11, or an amino acid sequence at least 90% identical thereto.

[0034] Thus, in a preferred embodiment, the modified signal peptide of the present invention comprises a region consisting of the amino acid sequence of any one of SEQ ID NOs: 7 to 12, or an amino acid sequence at least 90% identical thereto, and a region consisting of X1X2X3X4X5X6 adjacent to the C-terminus thereof. In a more preferred embodiment, the modified signal peptide of the present invention comprises the amino acid sequence of any one of SEQ ID NOs: 31 to 35.

[0035] The modified signal peptide of the present invention can be produced by modifying the C-terminal region of a parent signal peptide according to a genetic engineering technique known in the art. For example, individual amino acid residues in the C-terminal region of a parent signal peptide can be mutated (deleted, substituted, added, or inserted) to construct X1X2X3X4X5X6, or the C-terminal region of the parent signal peptide can be replaced with the amino acid sequence X1X2X3X4X5X6. Alternatively, the region from the C-terminus to the 7th to the N-terminus of the parent signal peptide can be bound to X1X2X3X4X5X6. In this case, the amino acid residues in the region from the C-terminus to the 7th to the N-terminus of the parent signal peptide can be mutated as long as the function as a signal peptide is not lost.

[0036] Preferably, the modified signal peptide of the present invention can be obtained by preparing a polynucleotide encoding the modified signal peptide and expressing it. The polynucleotide encoding the modified signal peptide can be prepared, for example, by mutating a polynucleotide encoding a parent signal peptide (hereinafter referred to as a parent gene) so as to encode the modified signal peptide. Alternatively, the nucleotide sequence encoding the modified signal peptide can be chemically synthesized.

[0037] The polynucleotide of the parent gene can be prepared by extracting genomic DNA from a strain that produces the parent signal peptide (e.g., Bacillus sp. KSM-S237 strain) using a standard method, or by extracting RNA and synthesizing cDNA by reverse transcription.

[0038] Means for mutating a parent gene include various mutagenesis techniques known in the art, such as ultraviolet irradiation, site-directed mutagenesis, splicing by overlap extension (SOE)-PCR (Gene, 1989, 77(1):p61-68), homologous recombination, etc. Commercially available site-directed mutagenesis kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit, etc.) can also be used.

[0039] Examples of polynucleotides encoding the modified signal peptide of the present invention include polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 36 to 41 or nucleotide sequences at least 90% identical thereto, and a nucleotide sequence encoding X1X2X3X4X5X6 linked downstream thereof. Preferred examples include polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 36 to 41 or nucleotide sequences at least 90% identical thereto, and a nucleotide sequence of SEQ ID NOs: 42 to 59 linked downstream thereof. More preferred examples include polynucleotides consisting of the nucleotide sequence of SEQ ID NOs: 39 or 40 or nucleotide sequences at least 90% identical thereto, and a nucleotide sequence of SEQ ID NOs: 42 to 59 linked downstream thereof. Further preferred examples include polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 60 to 64.

[0040] The modified signal peptide of the present invention can be used for the extracellular secretion of a target protein. For example, a polynucleotide encoding the modified signal peptide and a polynucleotide encoding a target protein are operably linked to express a preproprotein of the target protein having a signal peptide. The target protein is secreted extracellularly via the action of the modified signal peptide in the preproprotein.

[0041] Thus, the present invention provides a nucleic acid construct comprising a polynucleotide encoding a protein of interest and a polynucleotide encoding a modified signal peptide of the present invention linked upstream of the polynucleotide.

[0042] Preferably, the nucleic acid construct of the present invention is a nucleic acid construct for expressing a target protein in a host cell and secreting it outside the cell. Preferably, the nucleic acid construct of the present invention is an expression cassette, and includes a control region for controlling the expression of a polynucleotide encoding the target protein and a polynucleotide encoding a modified signal peptide. In the expression cassette, the polynucleotide encoding the target protein and the polynucleotide encoding the modified signal peptide are operably linked to the control region. Preferably, the expression cassette includes a promoter linked upstream of the polynucleotide encoding the modified signal peptide.

[0043] Preferred examples of the control region include, but are not limited to, control regions that function in bacteria of the genus Bacillus, for example, the control region of an α-amylase gene, a protease gene, an aprE gene, or a spoVG gene derived from a bacterium of the genus Bacillus, the control region of the cellulase gene of Bacillus sp. KSM-S237 strain (Patent Document 3), the control region of the cellulase gene of Bacillus sp. KSM-64 strain (JP Patent Publication No. 2011-10387), and the control region of a kanamycin resistance gene or a chloramphenicol resistance gene derived from Staphylococcus aureus (for both, see JP Patent Publication No. 2009-089708). More preferred examples of the control region include promoters derived from the genus Bacillus, such as the cellulase gene promoter of Bacillus sp. KSM-S237 strain (SEQ ID NO: 65), the cellulase gene promoter of Bacillus sp. KSM-64 strain (SEQ ID NO: 66), and the Bacillus subtilis spoVG gene promoter (SEQ ID NO: 67). Further, preferred control regions include promoters consisting of a nucleotide sequence having at least 90% identity with any of SEQ ID NOs: 65 to 67.

[0044] The nucleic acid construct of the present invention may contain a polynucleotide encoding the target protein and modified signal peptide, as well as a nucleic acid sequence other than the control region, such as a sequence of a 3' untranslated region or a 5' untranslated region. The nucleic acid construct may have a restriction enzyme recognition site at one or both ends. The nucleic acid construct of the present invention can be introduced into a vector using the restriction enzyme recognition site. For example, the vector can be cleaved with a restriction enzyme, and the nucleic acid construct of the present invention having a restriction enzyme recognition site at its end can be added thereto, thereby introducing the nucleic acid construct into the vector.

[0045] The type of target protein encoded in the nucleic acid construct of the present invention is not particularly limited. The target protein may be a heterologous protein, a homogeneous protein introduced from the outside, or a protein that the host cell can naturally express. Examples of target proteins include enzymes involved in the synthesis of any protein or target substance, such as antibody-related molecules, cytokines, hormones, other physiologically active peptides, transporters, industrial enzymes such as proteases, and metabolic enzymes.

[0046] Preferably, the target protein is an antibody-related molecule, more preferably a small antibody such as Fab, ScFv, or VHH. More preferably, the target protein is selected from the group consisting of Fab, ScFv, and VHH, more preferably VHH. Examples of Fab, ScFv, and VHH include, but are not limited to, those listed in Table 1 below.

[0047] [Table 1]

[0048] If necessary, the polynucleotide encoding the target protein contained in the nucleic acid construct of the present invention may be codon-optimized according to the species of the host into which the nucleic acid construct is to be incorporated. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0049] The nucleic acid construct of the present invention can be introduced into a host cell and used for secretory production of a target protein. The nucleic acid construct may be directly incorporated into the genomic DNA of the host cell, or may be incorporated into a vector and introduced into the host cell. The type of the vector is not particularly limited, and may be any vector such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, shuttle vector, etc. The vector may be a vector that is introduced into the genomic DNA of the host cell, or a vector that is maintained outside the genomic DNA. A vector that can replicate in a host cell is preferred. In one embodiment, the vector into which the nucleic acid construct is to be incorporated may be an expression vector, while in the case where the nucleic acid construct to be incorporated is an expression cassette, it does not have to be an expression vector. In one embodiment, the nucleic acid construct of the present invention is an expression cassette containing a control region, and is incorporated into any vector to construct an expression vector. In another embodiment, the nucleic acid construct of the present invention is incorporated into an expression vector containing a control region, and the expression cassette of the present invention is constructed on the expression vector.

[0050] Examples of preferred vectors 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), and pAC3 (Nucleic Acids Res, 1988, 16:8732); pUB110 (J Bacteriol, 1978, 134:318-329), pTA10607 (Plasmid, 1987, 18:8-15); and plasmids usable for transformation of bacteria of the genus Bacillus, such as pHY-S237 (JP Patent Publication No. 2014-158430). Plasmids derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0051] The type of host cell is not limited as long as the signal peptide of the present invention functions to secrete the target protein in the cell. A preferred host includes bacteria of the genus Bacillus, and more preferably Bacillus subtilis or a mutant thereof. A preferred example of a Bacillus subtilis mutant used as a host includes the extracellular protease-deficient Bacillus subtilis strain Dpr9 (see Japanese Patent No. 4485341). In addition, the Bacillus subtilis mutant used as a host may be subjected to deletion of sigma factors 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 obtained by adding deletion of one or both of sigF and recA to the Dpr9 strain may also be used.

[0052] The nucleic acid construct of the present invention or a vector containing the construct can be introduced into a host cell according to a standard method. For example, known transformation techniques such as the competent cell method, electroporation method, protoplast method, particle gun method, and PEG method can be applied to introduce the nucleic acid construct or vector into a host cell. The resulting recombinant cell containing the nucleic acid construct of the present invention can be used for secretion and production of a target protein.

[0053] A target protein can be produced by culturing a recombinant cell containing the nucleic acid construct of the present invention. The recombinant cell may be cultured according to a general culture method for the host cell. For example, a medium for culturing a Bacillus bacterium contains a carbon source and a nitrogen source necessary for the growth of the bacterium. If necessary, the medium may contain other nutrients such as inorganic salts, vitamins, antibiotics, etc. Culture conditions such as temperature, aeration and agitation conditions, pH of the medium, and culture time can be appropriately selected depending on the type and characteristics of the bacterium, the culture scale, etc.

[0054] By culturing the recombinant cell, a preproprotein of the target protein is expressed in the cell. The preproprotein contains the modified signal peptide of the present invention and a polypeptide encoding the target protein linked to its C-terminus. In the preproprotein, the modified signal peptide of the present invention is operably linked to the target protein, and the target protein is secreted outside the cell via the action of the modified signal peptide. Therefore, in the production of the target protein according to the present invention, it is not necessary to destroy the cells, and it is sufficient to collect the target protein secreted outside the cell. The collected target protein can be further purified by using a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination.

[0055] The target protein secreted outside the cell using the modified signal peptide of the present invention has a correct N-terminus, i.e., there are almost no unnecessary residues, such as signal peptide residues, remaining at the N-terminus. Therefore, the target protein produced by the present invention has little variation in structure and properties due to the unnecessary residues, and can well retain the function expected of the target protein. This is very advantageous for proteins whose activity is affected by slight structural differences, such as antibody-related molecules. For example, the antibody-related molecules produced by the present invention have no variation in activity and are highly valuable as raw materials for various medicines and reagents.

[0056] The present invention also includes the following substances, manufacturing methods, uses, methods, etc. as exemplary embodiments, but the present invention is not limited to these embodiments.

[0057] [1] A gene encoding a target protein, comprising a polynucleotide encoding a modified signal peptide linked upstream of the polynucleotide, The modified signal peptide has 1 to 6 amino acid residues from the C-terminus of X1X2X3X4X5X6, where X1 is G, P or A, X2 is G, S or A, X3 is P, S or A, with the proviso that X1X2X3 is not GGP, X4 is A, X5 is S, H or F, and X6 is A, and is located at the C-terminus of the modified signal peptide. A nucleic acid construct for expressing and secreting a protein of interest outside the cell. [2] Preferably, X1 is G, P or A, X2 is G, X3 is P or S, X4 is A, X5 is S or H, and X6 is A; More preferably, X1X2X3X4X5X6 consists of an amino acid sequence of any one of SEQ ID NOs: 13 to 30, More preferably, X1X2X3X4X5X6 consists of any one of the amino acid sequences of SEQ ID NOs: 15, 25 to 28. The nucleic acid construct described in [1]. [3] The region from the 7th residue from the C-terminus to the N-terminus of the modified signal peptide is Preferably, the amino acid sequence is an amino acid sequence of a region other than the C-terminal region of a signal peptide of the genus Bacillus, or an amino acid sequence at least 90% identical thereto, More preferably, it consists of an amino acid sequence of any one of SEQ ID NOs: 7 to 12, or an amino acid sequence that is at least 90% identical thereto, More preferably, it consists of the amino acid sequence of SEQ ID NO: 10 or 11, or an amino acid sequence at least 90% identical thereto. The nucleic acid construct according to [1] or [2]. [4] The nucleic acid construct according to [1], wherein the modified signal peptide preferably consists of an amino acid sequence of any one of SEQ ID NOs: 31 to 35. [5] The nucleic acid construct according to any one of [1] to [4], wherein the target protein is preferably an antibody-related molecule. [6] The antibody-related molecule, Preferably selected from the group consisting of Fab, ScFv and VHH, More preferably, it is a VHH. The nucleic acid construct described in [5]. [7] The nucleic acid construct according to any one of [1] to [6], preferably further comprising a promoter linked upstream of the polynucleotide encoding the modified signal peptide. [8] The promoter is Preferably, the promoter is derived from a bacillus bacterium. More preferably, it consists of any one of SEQ ID NOs: 65 to 67, or a nucleotide sequence having at least 90% identity thereto. The nucleic acid construct described in [7]. [9] The nucleic acid construct according to any one of [1] to [8], which is preferably a nucleic acid construct for expressing a target protein in a Bacillus cell and secreting it outside the cell.

[10] An expression vector comprising the nucleic acid construct according to any one of [1] to [9].

[11] A recombinant Bacillus bacterium comprising the nucleic acid construct according to any one of [1] to [9].

[0058]

[12] Cultivating the recombinant Bacillus bacterium according to

[11] ; and Recovering the target protein secreted outside the cell. A method for producing a target protein, comprising:

[13] The method according to

[12] , wherein the target protein is preferably an antibody-related molecule.

[14] The antibody-related molecule, Preferably selected from the group consisting of Fab, ScFv and VHH, More preferably, it is a VHH. The method described in

[13] .

[0059]

[15] A modified signal peptide, wherein the 1st to 6th amino acid residues from the C-terminus are X1X2X3X4X5X6, in which X1 is G, P or A, X2 is G, S or A, X3 is P, S or A, with the proviso that X1X2X3 is not GGP, X4 is A, X5 is S, H or F, and X6 is A, and the modified signal peptide is located at the C-terminus of the modified signal peptide.

[16] Preferably, X1 is G, P or A, X2 is G, X3 is P or S, X4 is A, X5 is S or H, and X6 is A; More preferably, X1X2X3X4X5X6 consists of an amino acid sequence of any one of SEQ ID NOs: 13 to 30, More preferably, X1X2X3X4X5X6 consists of any one of the amino acid sequences of SEQ ID NOs: 15, 25 to 28. The modified signal peptide described in

[15] .

[17] The region from the 7th residue from the C-terminus to the N-terminus is Preferably, the amino acid sequence is an amino acid sequence of a region other than the C-terminal region of a signal peptide of the genus Bacillus, or an amino acid sequence at least 90% identical thereto, More preferably, it consists of an amino acid sequence of any one of SEQ ID NOs: 7 to 12, or an amino acid sequence that is at least 90% identical thereto, More preferably, it consists of the amino acid sequence of SEQ ID NO: 10 or 11, or an amino acid sequence at least 90% identical thereto. The modified signal peptide according to

[15] or

[16] .

[18] The modified signal peptide according to

[15] , which preferably consists of any one of the amino acid sequences of SEQ ID NOs: 31 to 35.

[19] The modified signal peptide according to any one of

[15] to

[18] , which preferably functions in a bacterium of the genus Bacillus.

[0060]

[20] A preproprotein comprising the modified signal peptide according to any one of

[15] to

[19] above and a polypeptide encoding a target protein linked to the C-terminus thereof.

[21] The preproprotein according to

[20] , wherein the target protein is preferably an antibody-related molecule.

[22] The antibody-related molecule, Preferably selected from the group consisting of Fab, ScFv and VHH, More preferably, it is a VHH. The preproprotein described in

[21] . EXAMPLES

[0061] The present invention will be described in more detail below with reference to examples, although the technical scope of the present invention is not limited to these examples.

[0062] Materials and Methods (1) Host strain The host Bacillus subtilis was a derivative of Bacillus subtilis strain 168. 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), a sigF gene-deficient strain (Dpr9ΔsigF) was prepared according to the method described in Japanese Patent No. 4336082, and a recA gene-deficient strain (Dpr9ΔrecA) was prepared according to the method described in Japanese Patent No. 6088282. Furthermore, a Dpr9 strain in which sigF and recA were doubly deleted (Dpr9ΔsigFΔrecA) was prepared.

[0063] (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 necessary. DM3 medium: 1% CMC (Kanto Chemical), 0.5% Bacto TM Casamino Acids, 0.5% Bacto TM Yeast Extract, 8.1% disodium succinate 6H2O, 0.35% dipotassium hydrogen phosphate, 0.15% potassium dihydrogen phosphate, 0.5% glucose, 20 mM magnesium chloride, 0.01% BSA, 50 ppm tetracycline. 1% agar was added to the plate medium. L-mal medium: 1% Bacillus subtilis TM Tryptone, 0.5% Bacto TMYeast Extract, 0.5% Sodium Chloride, 3.75% Maltose Monohydrate, 3.75 ppm Manganese Sulfate, 15 ppm Tetracycline. 2×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.

[0064] (3) Reagents Unless otherwise specified, reagents used were manufactured by Wako.

[0065] Example 1 Construction of a plasmid containing a modified signal peptide (1) Artificial synthesis of genes The VHH genes (1ZVY and 5IMM, SEQ ID NOs: 68 and 69, respectively) were artificially synthesized by Thermo Fisher Scientific. A scaffold sequence GCAGCTCTTGCAGCA (SEQ ID NO: 70) was added to the 5' end of each of the artificially synthesized genes for inserting the genes into a plasmid vector, and TCTATTAAACTAGTT (SEQ ID NO: 71) was added to the 3' end of each of the artificially synthesized genes.

[0066] (2) Construction of plasmids for expressing antibody-related molecules (S237 signal peptide-1ZVY expression plasmid) The plasmid sequence was amplified by PCR using the plasmid pHY-S237 (JP Patent Publication 2014-158430 A) as a template and a primer set of SEQ ID NOs: 72 and 73 and PrimeSTAR Max DNA polymerase (TaKaRa). DNA containing the artificial synthetic gene of 1ZVY shown in (1) was inserted into the resulting PCR fragment using an In-Fusion HD Cloning Kit (Takara) to construct a plasmid for expressing 1ZVY containing the S237 signal peptide.

[0067] (yncM signal peptide-5IMM expression plasmid) The artificially synthesized gene of 5IMM was incorporated into pHY-S237 in the same manner as above. The resulting plasmid was used as a template, and a plasmid fragment was amplified by PCR using the primer set of SEQ ID NOs: 74 and 75 and PrimeSTAR Max DNA polymerase (TaKaRa). In addition, the genome of B. subtilis 168 strain was used as a template, and a promoter DNA (p_spoVG) derived from the spoVG gene was amplified by PCR using the primer set of SEQ ID NOs: 76 and 77. The p_spoVG DNA was incorporated into the amplified plasmid fragment using the In-Fusion HD Cloning Kit (Takara). The resulting plasmid was used as a template to amplify the plasmid sequence by PCR using the primer set of SEQ ID NOs: 78 and 79 and PrimeSTAR Max DNA polymerase (TaKaRa). In addition, the genome of B. subtilis 168 strain was used as a template to amplify the DNA of the yncM signal peptide by PCR using the primer set of SEQ ID NOs: 80 and 81. The DNA of the yncM signal peptide was integrated into the amplified plasmid sequence using the In-Fusion HD Cloning Kit (Takara) to construct a 5IMM expression plasmid containing the yncM signal peptide.

[0068] The primers used for plasmid construction are listed in Table 2.

[0069] [Table 2]

[0070] (3) Modification of the C-terminal region of the signal peptide The C-terminal region of the signal peptide on the plasmid constructed in (2) was modified using PrimeSTAR Mutagenesis Basal Kit (Takara) according to the manual. The C-terminal region of the obtained modified signal peptide consists of any one of the sequences of SEQ ID NOs: 13 to 30, and 82 to 84, as shown in Table 2 below. The modified plasmid fragment was treated with DpnI and then purified.

[0071] Example 2 Construction of recombinant Bacillus subtilis Plasmid introduction into the host Bacillus subtilis was performed by the protoplast method described below. The host Bacillus subtilis stocked in glycerol was inoculated into 1 mL of LB liquid medium and cultured overnight at 30°C and 210 rpm with shaking. The next day, 10 μL of this culture was inoculated into a new 1 mL of LB liquid medium and cultured for about 2 hours at 30°C and 210 rpm with shaking. This culture was collected in a 1.5 mL tube, centrifuged at 12,000 rpm for 5 minutes, and the pellet from which the supernatant was removed was suspended in 500 μL of SMMP containing 4 mg / mL of Lysozyme (SIGMA) and incubated at 37°C for 1 hour. Next, the mixture was centrifuged at 3,500 rpm for 10 minutes, and the pellet from which the supernatant was removed was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with 6 μL of the plasmid fragment constructed in Example 1, and 100 μL of 40% PEG was added and vortexed. To this solution, 350 μL of SMMP was added, mixed by inversion, and shaken at 30°C and 210 rpm for 1 hour, after which the entire amount was spread onto a DM3 agar medium plate and incubated at 30°C for 2 to 3 days. The recombinant Bacillus subtilis hosts prepared and the antibody-related molecules (VHH) and signal peptides encoded by the introduced plasmids are shown in Table 3. Note that for the plasmid containing the unmodified S237 signal peptide, the Dpr9ΔsigF strain into which this was introduced did not produce VHH, so the Dpr9ΔsigFΔrecA strain was used as the host to improve productivity.

[0072] [Table 3]

[0073] Test Example 1 Secretory production of VHH using modified S237 signal peptide (1) VHH production volume The recombinant Bacillus subtilis expressing 1ZVY (ID: 1-21) having the modified S237 signal peptide prepared in Example 2 was cultured in a 96-well plate. Proteins in the culture supernatant were collected, and the protein amount was quantified by SDS-PAGE. Detailed procedures for plate culture and protein quantification will be described later. The average protein amount (N=3) in the culture supernatant for each recombinant Bacillus subtilis is shown in Table 4. Secretion of VHH from recombinant Bacillus subtilis was successful with 18 of the 21 types of modified S237 signal peptides.

[0074] [Table 4]

[0075] (2) Analysis of the N-terminal amino acids of VHH The recombinant Bacillus subtilis (ID: 13 to 16) expressing 1ZVY having the modified S237 signal peptide prepared in Example 2 was cultured in a flask. 1ZVY was purified from the culture supernatant using a His-tag, and the N-terminal amino acid sequence of the obtained 1ZVY was analyzed. Detailed procedures for flask culture, His-tag purification, and analysis of the N-terminal amino acid sequence are described below. The N-terminal amino acid sequences of 1ZVY secreted into the culture supernatant were all DVQLV, and were completely identical to the N-terminus of the introduced 1ZVY.

[0076] (3) N-terminal amino acid analysis of VHH produced using unmodified S237 signal peptide In the same manner as in (2), the recombinant Bacillus subtilis (ID: Cnt-1) expressing 1ZVY having the unmodified S237 signal peptide prepared in Example 2 was cultured in a flask, 1ZVY was purified from the culture supernatant using a His-tag, and the N-terminal amino acid sequence of the obtained 1ZVY was analyzed. The N-terminal amino acid sequence of 1ZVY secreted into the culture supernatant was ADVQL, confirming that an unnecessary A derived from the signal peptide remained at the N-terminus of the secreted 1ZVY.

[0077] Test Example 2 Secretory production of VHH using modified yncM signal peptide (1) VHH production volume The 5IMM-expressing recombinant Bacillus subtilis (ID: 22-27) having the modified yncM signal peptide prepared in Example 2 was cultured in a 96-well plate. Proteins in the culture supernatant were collected, and the protein amount was quantified by SDS-PAGE. Detailed procedures for plate culture and protein quantification will be described later. The average protein amount (N=3) in the culture supernatant for each recombinant Bacillus subtilis is shown in Table 5. Secretion of VHH from recombinant Bacillus subtilis was successful with all six types of modified yncM signal peptides.

[0078] [Table 5]

[0079] (2) Analysis of the N-terminal amino acids of VHH The recombinant Bacillus subtilis (ID: 23) expressing 5IMM having the modified yncM signal peptide prepared in Example 2 was cultured in a flask. 5IMM was purified from the culture supernatant using a His-tag, and the N-terminal amino acid sequence of the resulting 5IMM was analyzed. Detailed procedures for flask culture, His-tag purification, and analysis of the N-terminal amino acid sequence are described below. The N-terminal amino acid sequences of all 5IMM secreted into the culture supernatant were QVQLV, which was completely identical to the N-terminus of the introduced 5IMM.

[0080] (3) N-terminal amino acid analysis of VHH produced using the unmodified yncM signal peptide In the same manner as in (2), the recombinant Bacillus subtilis (ID: Cnt-2) expressing 5IMM having the unmodified yncM signal peptide prepared in Example 2 was cultured in a flask, 5IMM was purified from the culture supernatant by His-tag purification, and the N-terminal amino acid sequence of the obtained 5IMM was analyzed. The N-terminal amino acid sequence of 5IMM secreted into the culture supernatant was TAXLF, and the third residue X from the N-terminus was Q for candidate residue 1, D for candidate residue 2, and V for candidate residue 3. When X was V, it matched the 23rd to 27th residues of the unmodified yncM signal peptide, TAVLF, confirming that unnecessary amino acid sequences derived from the signal peptide remained at the N-terminus of the secreted 5IMM.

[0081] Reference: Methods used in Test Examples 1 and 2 (1) Cultivation of recombinant Bacillus subtilis (96-well plate culture) The recombinant Bacillus subtilis prepared in Example 2 was inoculated into 300 μL of LB medium containing 50 ppm tetracycline and cultured overnight with shaking at 30° C. to prepare a preculture solution. The preculture solution was inoculated at 1% into 200 μL of L-mal medium placed in a 96-well plate and cultured with shaking at 30° C. for 72 hours. After the culture, 150 μL of the culture solution was centrifuged at 3,000 rpm at 4° C. for 10 minutes, and the supernatant was collected. (Flask culture) The recombinant Bacillus subtilis prepared in Example 2 was inoculated into 1 mL of LB medium containing 50 ppm tetracycline and shaken reciprocally overnight at 30° C. to prepare a preculture solution. The preculture solution was inoculated at 1% into 20 mL of 2×L-mal medium placed in a pleated Erlenmeyer flask and cultured with shaking at 30° C. for 72 hours. After the culture, 1 mL of the culture solution was centrifuged in a microtube at 4° C. and 15,000 rpm for 5 minutes, and the supernatant was collected.

[0082] (2) SDS-PAGE The culture supernatant collected in (1) was mixed with equal amounts of Laemmli Sample Buffer (BIO-RAD) and then heated at 99°C for 5 minutes to prepare the 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 the molecular weight marker. Protein bands were detected using a ChemiDoc MP Imaging System. The detected protein was quantified using a calibration curve created based on a lysozyme standard (Sigma-Aldrich).

[0083] (3)His-tag purification 50μL of Ni-NTA Agarose (Wako) was added to 1mL of the culture supernatant collected in (1), and the mixture was mixed by inversion on a rotator at 4℃ for 30 minutes. After centrifugation at 500g for 5 minutes, the supernatant was removed. 500μL of Wash Buffer (30mM imidazole, 0.5M NaCl, 10mM Tris, pH8.0) was added to the remaining precipitate, and the mixture was suspended by vortexing, then centrifuged at 500g for 5 minutes, and the supernatant was removed. This operation was repeated twice in total. 50μL of Elution Buffer (500mM imidazole, 0.5M NaCl, 10mM Tris, pH7.5) was added to the remaining precipitate, and the mixture was mixed by inversion on a rotator at 4℃ for 30 minutes. After centrifugation at 500g for 5 minutes, the supernatant was transferred to a new tube and used as a His-Tag purified sample.

[0084] (4) N-terminal amino acid analysis The purified sample obtained in (3) was mixed with an equal amount of Laemmli Sample Buffer (BIO-RAD) and then heated at 99°C for 5 minutes. Mini-PROTEIN TGX (BIO-RAD) was used for the gel. 10 μL of sample was applied to each well and electrophoresed at 210 V for 25 minutes. Proteins were transferred to a PVDF membrane using Trans-Blot Turbo Mini PVDF Transfer Packs (BIO-RAD) and Trans-Blot Turbo System (BIO-RAD), and stained with Bio-Safe CBB G-250 stain (BIO-RAD) for 30 minutes. The background was bleached using a 50% methanol solution, and the PVDF membrane was thoroughly washed with water, after which a band that matched the molecular weight of VHH was excised. The excised band was used as a sample to analyze the amino acid sequence of 5 residues from the N-terminus of VHH. Analysis of the amino acid sequence was entrusted to the Japan Leather Research Institute, a general incorporated foundation.

[0085] Although the embodiments of the present invention have been described above, it should be understood that they are not intended to limit the present invention to the specific embodiments described. Various other changes and modifications within the scope of the present invention will be apparent to those skilled in the art. The literature and patent applications cited in this specification are incorporated by reference as if fully set forth herein.

Claims

1. The present invention comprises a polynucleotide encoding a target protein and a polynucleotide encoding a modified signal peptide linked upstream of the polynucleotide, The modified signal peptide is as follows: the region from the 7th residue from the C-terminus to the N-terminus consists of the amino acid sequence of SEQ ID NO: 10 or 11, or an amino acid sequence at least 90% identical thereto; The 1st to 6th amino acid residues from the C-terminus are X 1 X 2 X 3 X 4 X 5 X 6 where X 1 is G, P or A, and X 2 is G and X 3 is P, S or A, where X 1 X 2 X 3 is not GGP, but X 4 is A and X 5 is S, H or F, and X 6 is A and is located at the C-terminus of the modified signal peptide; and having a function of secreting the target protein outside the cell; A nucleic acid construct for expressing and secreting a protein of interest outside the cell.

2. X 1 is G, P or A, and X 2 is G and X 3 is P or S, provided that X 1 X 2 X 3 is not GGP, and X 4 is A and X 5 is S, H or F, and X 6 The nucleic acid construct of claim 1 , wherein:

3. The nucleic acid construct according to claim 1, wherein X1, X2, X3, X4, X5, and X6 are each selected from the group consisting of an amino acid sequence of any one of SEQ ID NOs: 13 to 30.

4. X 1 X 2 X 3 X 4 X 5 X 6 The nucleic acid construct according to claim 1, wherein the nucleic acid construct comprises any one of the amino acid sequences of SEQ ID NOs: 15, 25 to 28.

5. The nucleic acid construct according to claim 1, wherein the modified signal peptide consists of an amino acid sequence of any one of SEQ ID NOs: 31 to 35.

6. The nucleic acid construct according to any one of claims 1 to 5, wherein the target protein is an antibody-related molecule.

7. The nucleic acid construct of claim 6 , wherein the antibody-related molecule is selected from the group consisting of Fab, ScFv and VHH.

8. The nucleic acid construct of claim 6 , wherein the antibody-related molecule is a VHH.

9. The nucleic acid construct according to any one of claims 1 to 8, further comprising a promoter linked upstream of the polynucleotide encoding the modified signal peptide.

10. The nucleic acid construct according to claim 9, wherein the promoter consists of any one of SEQ ID NOs: 65 to 67, or a nucleotide sequence having at least 90% identity thereto.

11. An expression vector comprising the nucleic acid construct according to any one of claims 1 to 10.

12. A recombinant Bacillus bacterium comprising the nucleic acid construct according to any one of claims 1 to 10.

13. Cultivating the recombinant Bacillus bacterium according to claim 12; and Recovering the target protein secreted outside the cell. A method for producing a target protein, comprising:

14. A modified signal peptide and a polypeptide encoding an antibody-related molecule linked to the C-terminal side thereof, The modified signal peptide is as follows: The 1st to 6th amino acid residues from the C-terminus are X 1 X 2 X 3 X 4 X 5 X 6 where X 1 is G, P or A, and X 2 is G and X 3 is P, S or A, where X 1 X 2 X 3 is not GGP, but X 4 is A and X 5 is S, H or F, and X 6 is A and is located at the C-terminus of the modified signal peptide; The amino acid sequence from the 7th amino acid from the C-terminus to the N-terminus consists of the amino acid sequence of SEQ ID NO: 10 or 11, or an amino acid sequence at least 90% identical thereto; and and having a function of secreting the polypeptide encoding the antibody-related molecule outside the cell. A preproprotein of an antibody-related molecule.

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