Acinetobacter bacterium and method for producing protein using the same

Abstract

To provide a bacterium serving as a host in production of a target protein, in which contamination with endotoxin and the like is inhibited.SOLUTION: A bacterium belonging to the genus Acinetobacter has an inhibited function of a lipopolysaccharide biosynthetic protein and of a BaeR protein and / or a BaeS protein.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to bacteria belonging to the genus Acinetobacter (Acinetobacter bacteria) and a method for producing a protein using the same. More specifically, the present invention relates to Acinetobacter bacteria in which the functions of lipopolysaccharide (LPS) biosynthesis protein and BaeR protein and / or BaeS protein are suppressed, and a method for producing a target protein in which contamination such as endotoxin is suppressed using the same.

Background Art

[0002] An expression system for a target foreign protein using bacteria (recombinant protein expression system) is an indispensable tool in modern science and biopharmaceutical production. Although using bacteria as an expression host cannot be applied to some products due to the lack of sugar chains, the production cost can be significantly reduced compared to cultured cell lines. In particular, Escherichia coli (E. coli) is a Gram-negative bacterium that has been used as a model organism since the dawn of molecular biology and has established a firm position in protein production. Many tools are available for protein expression in E. coli compared to other bacteria and cultured cell lines.

[0003] However, lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria such as E. coli is also known as an endotoxin, and its contamination is an important concern in recombinant protein expression systems using bacteria. Endotoxin can cause an inflammatory response in mammals and ultimately lead to septic shock. Therefore, it is essential to completely remove endotoxin in order to safely administer biopharmaceuticals expressed in E. coli to human patients.

[0004] Various attempts have been made to remove endotoxins from biopharmaceuticals and the like. For example, by genetically engineering the BL21(DE3) strain of E. coli, a strain called ClearColi was created that alleviates the problem of endotoxin contamination. In this strain, the original hexaacyl-type lipid A of LPS is converted to tetraacyl-type lipid A (lipid IVA), thereby suppressing the endotoxin reaction in humans (Non-Patent Documents 1 to 4). Furthermore, as hosts for protein expression instead of Gram-negative bacteria containing LPS, methods using fungi and Gram-positive bacteria such as yeast, Bacillus subtilis, and Brevibacillus (Brevibacillus brevis, Brevibacillus choshinensis) have been investigated (Non-Patent Documents 5 to 7). However, it should be noted that all of these methods are positive in the LAL assay, which is the most sensitive and reliable method for detecting bacterial endotoxins. Even if it is a commercially produced recombinant protein derived from E. coli, there is a possibility that it contains trace amounts of endotoxin (Non-Patent Document 8).

[0005] The LAL assay is a method for detecting bacterial endotoxins that uses extracts of horseshoe crab blood cells. This method was approved by the US Food and Drug Administration (FDA) in the 1970s and is designated by the Pharmacopoeia Discussion Group (PDG) as the only endotoxin test method in Japan, the US, and Europe.

[0006] As described above, the removal of endotoxins from biopharmaceuticals requires great efforts and significantly increases the development and manufacturing costs (Non-Patent Document 9). However, a method for producing a recombinant protein that is substantially free of endotoxins has not been established to date (Non-Patent Documents 10 to 12).

[0007] In recent years, biopharmaceuticals such as low-molecular-weight antibodies have attracted attention as a new drug discovery modality. For example, VHH (variable domain of heavy-chain antibody), also known as nanobody, can be easily modified into multivalent antibodies, bispecific antibodies, antibody-drug conjugates (ADCs), etc. using protein engineering techniques, and thus has great potential for commercial applications in the medical and diagnostic fields. In addition, VHH antibodies are more stable than ordinary antibodies and retain their activity even under harsh conditions such as temperature and pH fluctuations. Furthermore, since the molecular weight of VHH antibodies is very small (about 15 kDa), they can be produced even using bacteria.

[0008] Thus, it is possible to express low-molecular-weight antibodies even in bacteria. However, inclusion body formation is often observed, and complicated operations such as refolding are required to obtain active low-molecular-weight antibodies from them. Also, as mentioned above, bacterial endotoxin contamination becomes an issue. In particular, since E. coli has endotoxin in its outer membrane, its application as a production host for biopharmaceuticals is greatly restricted (Non-Patent Documents 4, 13, and 14). Therefore, many biopharmaceuticals are produced using cultured mammalian cells or insect cells, which significantly increases the production cost (Non-Patent Document 15).

[0009] Therefore, in the production of biopharmaceuticals, etc., the establishment of a system using bacteria that enables the expression of target proteins substantially free of endotoxin is desired. However, such an expression system and useful bacteria for it have not yet been developed.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present invention has been made in view of the problems of the prior art, and an object thereof is to provide a bacterium serving as a host in which contamination with endotoxin is suppressed in the production of a target protein.

Means for Solving the Problems

[0012] In Acinetobacter baumannii (A. baumannii), strains resistant to drugs targeting LPS (e.g., colistin) exhibit an interesting phenotype of complete LPS deficiency and reduced pathogenicity (Non-Patent Documents 16 and 17). Also, although LPS is usually regarded as an essential component for the survival of Gram-negative bacteria, A. baumannii has a unique property that it can survive even after completely losing LPS by introducing mutations in the lpxA / C / D genes involved in the early stage of LPS biosynthesis (Non-Patent Documents 18 to 20).

[0013] The inventors of the present invention focused on such a unique property of A. baumannii and conducted intensive research to achieve the above object. First, by using an LPS-deficient strain of A. baumannii (lpxC gene mutant, KL037S strain disclosed in Non-Patent Document 18), we succeeded in producing cytokines such as green fluorescent protein (GFP) and tumor necrosis factor (TNF)-α while keeping the contamination of endotoxin extremely low and maintaining the functions of these proteins.

[0014] Furthermore, by using a periplasmic translocation signal peptide, it was found that extracellular secretion production can be performed more efficiently, and it was also clarified that the above LPS-deficient strain of A. baumannii is useful in the production of low-molecular antibodies, which have been difficult to produce so far.

[0015] On the other hand, in the protein production in the defective strain, contamination with non-specific proteins was sometimes observed. Therefore, as a result of creating a further mutant strain of the KL037S strain, we succeeded in obtaining a mutant strain (Eep2 strain) in which the expression of non-specific proteins was suppressed. Furthermore, it was also found that in the mutant strain, the growth ability, which was reduced in the KL037S strain, was improved.

[0016] In addition, as a result of performing mutation detection on the Eep2 strain by next-generation sequencing (NGS; Next-Generation Sequencing), mutations that can induce functional suppression of the proteins encoded by each gene were observed in three genes, namely, the baeR gene (Locus_tag: KAMO5_05630), the polyhydroxyalkanoate granule-binding protein (Phasin) family protein gene (Locus_tag: KAMO5_18120), and the phosphatidic acid phosphatase type 2 (PAP2) family protein gene (Locus_tag: KAMO5_07110), when compared with the parental strain. Furthermore, as a result of analyzing transformants of the KL037S strain in which contamination with non-specific proteins was suppressed, mutations were observed in the gene encoding BaeS, which is a counterpart of BaeR.

[0017] Furthermore, in an attempt to improve the decrease in growth associated with LPS deficiency, first, an artificial LPS-deficient strain was prepared by disrupting the lpxA gene in the ATCC19606 strain, which is the parental strain of the KL037S strain. Then, based on the LPS-deficient strain, a combined artificial-deficient strain of the three genes (baeR, PAP2, phasin) in which disruption was observed with Eep2 was prepared, and the growth ability was evaluated. As a result, the growth ability, which was significantly decreased due to lpxA deficiency, was partially improved by adding baeR gene deficiency, and further significant improvement in growth ability was observed by adding PAP2 gene deficiency, leading to the completion of the present invention.

[0018] That is, the present invention provides the following aspects.

[0019] [1] A bacterium belonging to the genus Acinetobacter in which the function of a lipopolysaccharide (LPS) biosynthesis protein is suppressed.

[0020] [2] The bacterium according to [1], wherein the LPS biosynthesis protein is at least one protein selected from the group consisting of an LpxC protein, an LpxA protein, and an LpxD protein.

[0021] [3] The bacterium according to [1] or [2], further comprising a suppressed function of the BaeR protein and / or the BaeS protein.

[0022] [4] The bacterium according to any one of [1] to [3], further comprising a suppressed function of a phosphatidic acid phosphatase type 2 (PAP2) family protein.

[0023] [5] The bacterium according to any one of [1] to [3], further comprising a suppressed function of a polyhydroxyalkanoate granule-associated protein (Phasin) family protein and / or a phosphatidic acid phosphatase type 2 (PAP2) family protein.

[0024] [6] The bacterium according to any one of [1] to [5], wherein the bacterium belonging to the genus Acinetobacter is Acinetobacter baumannii.

[0025] [7] The bacterium according to any one of [1] to [6], into which a DNA construct capable of expressing a target protein has been introduced.

[0026] [8] The bacterium according to [7], wherein the target protein is a fusion protein with a histidine tag peptide.

[0027] [9] The bacterium described in [7] or [8], wherein the DNA construct is a DNA construct capable of expressing the target protein by fusing a periplasmic transport signal peptide to the N-terminus of the target protein.

[0028]

[10] The bacterium described in [9], wherein the periplasmic transport signal peptide is a signal peptide derived from the Omp38 protein.

[0029]

[11] A method for producing a target protein, comprising: A step of culturing the bacterium according to any one of [7] to

[10] ; and a step of isolating the target protein from the culture obtained in the step. A method including

[0030]

[12] A kit for producing a target protein, a bacterium according to any one of [1] to [6], and a DNA construct capable of expressing the target protein, the kit comprising.

[0031]

[13] The kit according to

[12] , wherein the target protein is a fusion protein with a histidine-tag peptide.

[0032]

[14] The kit according to

[12] or

[13] , wherein the DNA construct is a DNA construct capable of expressing by fusing a periplasmic translocation signal peptide to the N-terminus of the target protein.

[0033]

[15] The kit according to

[14] , wherein the periplasmic translocation signal peptide is a signal peptide derived from the Omp38 protein.

[0034]

[16] A DNA construct capable of expressing by fusing a signal peptide derived from the Omp38 protein to the N-terminus of the target protein.

Advantages of the Invention

[0035] According to the present invention, it becomes possible to produce a target protein substantially without including endotoxin. In particular, according to the present invention, it is possible to produce a target protein in which endotoxin contamination is almost completely eliminated to such an extent that it becomes a background level value in the LAL assay, which is the most sensitive and reliable method in the detection of bacterial endotoxin.

[0036] Further, according to the present invention, it becomes possible to efficiently grow bacteria serving as an expression host of a target protein and to produce the target protein while suppressing the expression and contamination of non-specific proteins.

[0037] Furthermore, according to the present invention, it is also possible to perform more efficient extracellular secretion production while maintaining the function of the target protein.

Brief Description of the Drawings

[0038]

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Figure 11

Mode for Carrying Out the Invention

[0039] <Bacteria> As shown in the examples described below, the present inventors have succeeded in producing a target protein such as a cytokine with extremely low contamination of endotoxin and maintaining its function by using, as a host, a bacterium belonging to the genus Acinetobacter lacking LPS by suppressing the function of lipopolysaccharide (LPS) biosynthetic protein.

[0040] Furthermore, the present inventors have found that by suppressing the function of BaeR and / or BaeS, the growth ability can be improved and the expression of non-specific proteins can be suppressed. Furthermore, it has been clarified that by suppressing the function of the phosphatidic acid phosphatase type 2 (PAP2) family protein, the growth ability is further improved.

[0041] Therefore, the present invention relates to the following bacteria.

[0042] A bacterium belonging to the genus Acinetobacter, in which the function of the LPS biosynthesis protein is suppressed, and further, the function of the BaeR protein and / or the BaeS protein may be suppressed, and furthermore, the function of the PAP2 family protein and / or the Phasin family protein may be suppressed.

[0043] In the present invention, "bacteria belonging to the genus Acinetobacter (Acinetobacter bacteria)", in which the functions of various proteins are suppressed, are eubacteria of Gram-negative bacilli, and mean bacteria belonging to the genus Acinetobacter of the family Moraxellaceae, Pseudomonadales, Gammaproteobacteria class, Proteobacteria phylum.Examples of such bacteria include Acinetobacter baumannii (A. baumannii), Acinetobacter baylyi (A. baylyi), Acinetobacter beijerinckii (A. beijerinckii), Acinetobacter bereziniae (A. bereziniae), Acinetobacter boissieri (A. boissieri), Acinetobacter bouvetii (A. bouvetii), Acinetobacter brisouii (A. brisouii), Acinetobacter calcoaceticus (A. calcoaceticus), Acinetobacter colistiniresistens (A. colistiniresistens), Acinetobacter gerneri (A. gerneri), Acinetobacter guillouiae (A. guillouiae), Acinetobacter grimontii (A. grimontii), Acinetobacter gyllenbergii (A. gyllenbergii), Acinetobacter haemolyticus (A. haemolyticus), Acinetobacter indicus (A. indicus), Acinetobacter johnsonii (A. johnsonii), Acinetobacter junii (A. junii), Acinetobacter lwoffii (A. lwoffii), Acinetobacter nectaris (A. nectaris), Acinetobacter nosocomialis (A. nosocomialis), Acinetobacter parvus (A. parvus), Acinetobacter pittii (A. pittii), Acinetobacter puyangensis (A. puyangensis), Acinetobacter radioresistens (A. radioresistens), Acinetobacter rudis (A. rudis), Acinetobacter schindleri (A. schindleri), Acinetobacter soli (A. soli), Acinetobacter tandoii (A. tandoii), Acinetobacter tjernbergiae (A. tjernbergiae), Acinetobacter towneri (A. towneri), Acinetobacter ursingii (A. ursingii), and Acinetobacter venetianus (A. venetianus).Among these, from the perspective of richer research findings, Acinetobacter baumannii and Acinetobacter nosocomialis are preferred, Acinetobacter baumannii is more preferred, and the standard strain ATCC19606 strain is even more preferred.

[0044] In the present invention, as a protein whose function is suppressed, there is a lipopolysaccharide biosynthesis protein. "Lipopolysaccharide (LPS)" is also referred to as endotoxin and is a component of the outer membrane of Gram-negative bacteria, mainly referring to a substance (glycolipid) composed of lipids and polysaccharides. "LPS biosynthesis protein" refers to proteins (mainly enzymes) involved in the biosynthesis of LPS, such as LpxA, LpxB, LpxC, LpxD, LpxK, LpxL, LpxH, LpxM, LpxO. In the present invention, in the early stage of the biosynthesis of lipid A, which is the active center of LPS, LpxA, LpxC, and LpxD act in this order. From the perspective that the functional deficiency of these enzymes is more likely to lead to the deficiency of LPS, it is preferable that the function of at least one protein selected from the group consisting of LpxA, LpxC, and LpxD is suppressed. In addition, among these lipopolysaccharide biosynthesis proteins, those derived from Acinetobacter bacteria are shown in Table 1 below.

[0045]

Table 1

[0046] In addition, in the present invention, from the perspective of improving the growth ability of the bacteria of the present invention and suppressing the expression of non-specific proteins, it is preferable to suppress the functions of BaeR and / or BaeS. Furthermore, from the perspective of further improving the growth ability of the bacteria of the present invention, it is preferable to suppress the function of the PAP2 family protein. Among these proteins, those derived from Acinetobacter bacteria are shown in Table 2 below. The "non-specific protein" according to the present invention mainly refers to a protein having a molecular weight of about 12 kDa and having binding properties to metal chelates (nickel, cobalt, copper, etc.), as shown in the examples described later.

[0047]

Table 2

[0048] The typical amino acid sequences of each protein are shown in Tables 1 and 2. However, the proteins according to the present invention are not limited to those specified by these typical sequences.

[0049] In nature, it is possible for nucleotide sequences to mutate. Accordingly, the amino acids encoded thereby may also change. Therefore, the proteins according to the present invention also include proteins (variants, mutants) consisting of amino acid sequences in which one or more amino acids are substituted, deleted, added, and / or inserted in the above-described typical wild-type amino acid sequences. With respect to the proteins according to the present invention, the term "a plurality" is not particularly limited. For example, it may be within 100 amino acids (within 90 amino acids, within 80 amino acids, within 70 amino acids, within 60 amino acids, etc.), within 50 amino acids (within 40 amino acids, within 30 amino acids, within 20 amino acids, etc.), within 10 amino acids (within 9 amino acids, within 8 amino acids, within 7 amino acids, within 6 amino acids, etc.), or within several amino acids (within 5 amino acids, within 4 amino acids, within 3 amino acids, within 2 amino acids).

[0050] In addition, the protein according to the present invention also includes a protein (homolog) having an amino acid sequence with high homology (high similarity), preferably high identity, to the above-described typical wild-type amino acid sequence. Here, "high" means at least 30% or more, preferably 40% or more, more preferably 50% or more, still at least 60% or more, more preferably 70% or more, still more preferably 80% or more, and even more preferably 85% or more (for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more). The sequence homology can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). For example, when analyzing an amino acid sequence by BLAST, the parameters are, for example, score = 50 and wordlength = 3. When analyzing an amino acid sequence using the Gapped BLAST program, it can be performed as described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

[0051] In the present invention, "suppression of protein function" is not particularly limited as long as the function can be suppressed or inhibited (completely suppressed). Examples include inhibition of synthesis, promotion of degradation, and inactivation of the protein. "Inhibition of synthesis" includes not only inhibition of protein synthesis (translation) based on mRNA, but also inhibition of mRNA synthesis (transcription) based on genomic DNA (gene) and mutations in the gene.

[0052] The site where the mutation is introduced into the gene is not particularly limited as long as the function can be suppressed. For example, it includes the coding region, non-coding region, expression control region (endogenous promoter region) of the gene, and the like.

[0053] In the present invention, the mutation introduced into the gene is not particularly limited as long as the function can be suppressed. For example, it includes nucleotide deletion, substitution, addition, and / or insertion. From the viewpoint that the introduced mutation is difficult to recover, it is desirable that it is a large nucleotide deletion (for example, a nucleotide deletion accompanied by a deletion of at least 10% of the amino acid sequence encoded by each of the genes described below). Further, the mutation according to the present invention may be a frameshift mutation, null mutation, nonsense mutation, or the like. Furthermore, the number of mutations introduced into each of the genes is not particularly limited as long as the function is suppressed, and it may be 1 or a plurality (for example, 2, 3 or less, 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less).

[0054] Such a mutation does not necessarily need to cause the loss of the entire amino acid sequence encoded by the gene, and it may be introduced into the gene so that a part of it is lost or changed. For example, a nucleotide mutation accompanied by a change or deletion of at least 10% (preferably 20% or more, more preferably 30% or more, still more preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, more preferably 90% or more, particularly preferably 100%) of the amino acid sequence encoded by each of the genes may be introduced.

[0055] The region where the amino acid sequence is changed or deleted in this way is not particularly limited as long as the function is suppressed. For example, those skilled in the art can grasp the functional domain in the amino acid sequence encoded by each gene based on information such as Uniprot, and thus can introduce a mutation targeting the nucleotide sequence encoding the domain.

[0056] As a mutation according to the present invention, from the viewpoint of being able to produce a target protein substantially without endotoxin, specifically, for example, 475_689(215)del:D159fs(D159C)D161* in the lpxC gene, 591_592delTA:I198fs(I198*) in the lpxA gene, 218insA: D75fs(D75R)N76* in the lpxD gene are cited as preferred mutation examples in each LPS biosynthesis gene (see Table 1 etc. of Non-Patent Document 18). Also, those skilled in the art can design mutations that can suppress the function of LPS biosynthesis proteins with reference to the mutation examples in each LPS biosynthesis gene disclosed in FIG. 1A of Non-Patent Document 18 and introduce them into bacteria of the genus Acinetobacter.

[0057] Here, "substantially free of endotoxin" can be evaluated by an assay using a horseshoe crab blood cell extract (LAL). Specifically, the (contamination) concentration of endotoxin in the target protein produced by the present invention is preferably 10 EU / mg or less, more preferably 7 EU / mg or less, still more preferably 5 EU / mg or less, more preferably 3 EU / mg or less, still more preferably 2 EU / mg or less, and even more preferably 1 EU / mg or less. Also, as bacteria substantially free of endotoxin, specifically, the endotoxin concentration of the bacterial suspension (optical density (OD600) at 600 nm is 0.1) is preferably 0.1 EU / mL or less, more preferably 0.07 EU / mL or less, still more preferably 0.05 EU / mL or less, more preferably 0.03 EU / mL or less, and still more preferably 0.02 EU / mL or less.

[0058] "Suppression of protein function" can be achieved by methods known to those skilled in the art. Such known methods include homologous recombination; genome editing methods; methods targeting transcripts using sRNA, siRNA, shRNA, antisense RNA, RNA having ribozyme activity, etc.; CRISPR gene transcriptional repression (CRISPRi), etc. Those skilled in the art can specifically suppress the function of the said protein by such methods.

[0059] "Homologous recombination" is not particularly limited as long as part or all of the said gene is deleted. For example, two-step homologous recombination by positive selection, counter selection, etc., as shown in the examples described later, can be mentioned. Also, gene disruption can be performed by single homologous recombination. Furthermore, for example, gene disruption by homologous recombination can also be performed using the lambda red recombination system (T. Baba et al., Mol. Syst. Biol., 2006, 2, 20060008), Cre / Lox, attB / attP, other integrase systems.

[0060] The "genome editing method" is a method of modifying a target gene using a site-specific nuclease (for example, DNA double-strand cleavage enzymes such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR-Cas9). For example, methods using fusion proteins such as ZFNs, TALENs, PPR fused with a nuclease domain, and complexes of guide RNA and protein such as CRISPR-Cas9, CRISPR-Cpf1, Target-AID can be mentioned.

[0061] Also, in the present invention, "suppression of protein function" may be a method involving not only the gene encoding the said protein but also introduction of mutations (non-specific mutation introduction) into other genes. Such known methods include physical mutation introduction methods, methods using chemical mutagens, methods of introducing transposable elements into genomic DNA, etc.

[0062] Examples of the "physical mutagenesis method" include heavy ion beam (HIB) irradiation, fast neutron beam irradiation, gamma ray irradiation, and ultraviolet irradiation.

[0063] Examples of the "method using a chemical mutagen" include a method of treating with a chemical mutagen. The chemical mutagen is not particularly limited, and examples thereof include ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, formic acid, and nucleotide analogs.

[0064] Examples of the "method of introducing a transposable element into genomic DNA" include the method described by H. Mori et al., Research in Microbiology, 2000, Vol. 151, No. 2, pp. 121-128. The transposable element is not particularly limited, and examples thereof include transposons and insertion sequence (IS) elements. More specifically, examples of transposons include Tn6021, Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn9, Tn10, Tn204, Tn402, Tn501, Tn551, Tn554, Tn732, Tn903, Tn917, Tn951, Tn1681, Tn1721, etc., and examples of IS elements include ISAba11, ISAcma22, IS4 family, IS1 family, IS3 family, IS150 family, IS911 family, etc.

[0065] Also, as shown in the examples described later, mutations can also be introduced into the genomic DNA of the bacterium by applying stimuli such as electroporation to the bacterium.

[0066] For the bacteria into which mutations have been introduced by the above method, it is possible to confirm, by known methods, that mutations have been introduced into genes encoding proteins involved in the biosynthesis of LPS and the like. Examples of such known methods include the DNA sequencing method (next-generation sequencing (NGS) method, etc.), the PCR method, the analysis method using a microarray, the Southern blot method, and the Northern blot method. According to such methods, it is possible to determine whether or not a mutation has been introduced into the gene by comparing the sequence or length of the gene before and after the introduction of the mutation. In addition, by using methods such as the Northern blot method, the RT-PCR method, the Western blot method, the ELISA method, and the analysis method using a microarray, if a decrease in the expression level of the transcription product or translation product of the gene (preferably, the expression level has substantially disappeared) is observed in the bacteria into which the mutation has been introduced into the gene, it can also be confirmed that the cell is a bacterium into which the mutation has been introduced into the gene.

[0067] In addition, as another method for confirming that a mutation has been introduced into the gene, TILLING (Targeting Induced Local Lesions IN Genomes) can be mentioned (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when non-specific mutations are introduced into the genome using the above-described heavy ion beam irradiation, chemical mutagens, insertion of transposons, etc., after amplifying the gene or a part thereof by PCR, cells having a mutation in the amplification product can be selected by the above-described TILLING or the like.

[0068] Furthermore, in the present invention, the suppression of the function of the protein may be not only by the introduction of artificial mutations as described above, but also by natural mutations.

[0069] <DNA construct> In the present invention, by introducing a DNA construct capable of expressing a target protein into the above-described bacterium, it becomes possible to produce the protein without including endotoxin or the like. Such a "DNA construct" can be constructed based on, for example, a plasmid, which is a self-replicating vector, that is, it exists as an extrachromosomal entity and its replication does not depend on chromosomal replication. Further, the DNA construct may be integrated into the genome of the bacterium of the present invention when introduced into the bacterium and replicated together with the chromosome into which it is integrated. Examples of such "plasmids" include pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series. Further, such a plasmid preferably has an origin of replication (Ori) that can be amplified at least in bacteria of the genus Acinetobacter in order to ensure self-replicating ability. Further, the DNA construct of the present invention may adopt a mode in which it can replicate in other biological systems different from bacteria of the genus Acinetobacter (that is, a shuttle vector). In such a case, for example, the DNA construct of the present invention may also have an origin of replication that can be amplified in other biological systems (such as Escherichia coli).

[0070] The DNA construct of the present invention also contains one or more regulatory elements operably linked to the DNA encoding the target protein for expressing the target protein. Here, "operably linked" means that the above DNA is expressibly linked to the regulatory element. Examples of "regulatory elements" include a promoter, a terminator, and a ribosome binding sequence (SD sequence).

[0071] The "promoter" is not particularly limited as long as it is a sequence capable of inducing the transcription of DNA operably linked downstream thereof in bacteria belonging to the genus Acinetobacter, and may be a constitutive promoter or an inducible promoter. The "constitutive promoter" may be any sequence capable of constantly and strongly inducing the transcription of operably linked DNA. More specifically, examples include the T5 promoter, T3 promoter, T7 promoter, TetA promoter, etc. The "inducible promoter" may be any sequence capable of inducing the transcription of operably linked DNA under predetermined conditions. Here, the "predetermined conditions" include the presence or absence of a compound (such as sugar, amino acid, peptide, protein, antibiotic, nutrient, metabolite, etc.), the presence or absence of a metal, the presence or absence of light irradiation, high temperature or low temperature, and the presence or absence of a gas (such as oxygen). Specific examples of the "inducible promoter" capable of inducing expression according to such conditions include the lac promoter induced by IPTG, lactose, allolactose, etc., the promoter of the Tet-on system / Tet-off system induced in the presence or absence of tetracycline or its derivative (such as doxycycline), the Trp promoter induced in the absence of tryptophan, the Tac promoter composed of a combination of the lac promoter and the Trp promoter, the GAL1 promoter and GAL10 promoter induced by galactose, etc., the araBAD promoter induced by arabinose, the CUP1 promoter induced by copper ions, etc. Further, among such inducible promoters, some have an operator sequence like the lac promoter. Under non-inducing conditions, the transcription is suppressed because a repressor (such as the lac repressor; LacI) binds to this operator sequence (for example, the lac operator). However, under inducing conditions, the transcription becomes possible because the repressor dissociates from the operator sequence. Therefore, the "inducible promoter" according to the present invention also includes a promoter further provided with an operator sequence.

[0072] As the "terminator", any sequence capable of terminating transcription by the promoter may be used. For example, the rrnB terminator (such as rrnB T1 terminator, rrnB T2 terminator, etc.) can be mentioned.

[0073] The DNA construct of the present invention may have an insertion site (cloning site) for operably linking DNA encoding a target protein. Examples of such "insertion sites" include a multiple cloning site, a TA cloning site, a TOPO cloning site, and a Gateway cloning site. Also, it may be a terminal DNA homologous region in seamless cloning such as In-Fusion and Gibson Assembly.

[0074] In order to express the target protein, the DNA encoding the protein is inserted into the insertion site in the DNA construct of the present invention. Note that the DNA may be optimized for codons suitable for the bacterium in order to efficiently express its translation product in bacteria belonging to the genus Acinetobacter. The "target protein" according to the present invention can also be referred to as a recombinant protein, a foreign protein, a desired protein, an arbitrary protein, etc., and is not particularly limited as long as it can be expressed in bacteria belonging to the genus Acinetobacter. Also, not only proteins in the narrow sense but also proteins in the broad sense, that is, peptides, oligopeptides, and polypeptides are included in the target protein of the present invention. The "target protein" according to the present invention is not particularly limited as described above, but examples include bioactive proteins (such as cytokines, growth factors, hormones, etc.), antibodies, antigens, enzymes, etc. Also, as the "antibody", as shown in the examples described later, small molecule antibodies are preferable as the production targets of the present invention. More specifically, VHH (nanobody), Fab, Fab', F(ab')2, variable region fragment (Fv), disulfide bond Fv, single-chain variable region fragment (single-chain Fv, scFv), sc(Fv)2, diabody, multispecific antibody, and polymers thereof can be mentioned.

[0075] The "target protein" of the present invention may also include other proteins. There are no particular restrictions on the "other proteins", and examples include purification tag peptides such as histidine (His) tag peptide, FLAG tag peptide (registered trademark, Sigma-Aldrich), glutathione-S-transferase (GST); detection tag proteins such as fluorescent proteins such as GFP and chemiluminescent proteins such as luciferase. In the target protein of the present invention, such other proteins are not particularly limited, but are usually arranged at the N-terminus and / or C-terminus. Further, by further including an enzyme recognition sequence and using the corresponding enzyme (blood coagulation factor Xa, TEV protease, thrombin, etc.), it becomes possible to exclude other proteins from the target protein.

[0076] Also, as shown in the examples described later, when the function of the LPS biosynthesis protein is suppressed and the target protein is expressed by fusing a periplasmic translocation signal in Acinetobacter bacteria, the protein does not remain in the periplasmic translocation but can be secreted extracellularly. Therefore, in the DNA construct of the present invention, a DNA encoding a periplasmic translocation signal peptide may be inserted near the insertion site so that the periplasmic translocation signal peptide can be fused to the N-terminus of the target protein for expression. By taking such an aspect, it becomes possible to secrete the target protein to the extracellular space, eliminating the need for cell disruption treatment in the purification process, and reducing costs associated with the disruption treatment and enabling reuse of the bacterial cells.

[0077] The "periplasmic translocation signal peptide" may be any peptide that translocates (secretes) the target protein fused to its C-terminal side into the periplasm. Examples include signal peptides of omp (ompA, ompF, ompT, etc.), pelB, phoA, lamB, malE, and dsbA. Since a protease recognition sequence exists on the C-terminal side of such a signal peptide, it is cleaved when passing through the inner membrane during periplasmic translocation, and the target protein is secreted into the culture solution or the like in a form in which the signal peptide is cleaved.

[0078] As the periplasmic translocation signal peptide according to the present invention, as shown in the examples described later, since it has the highest expression level at the mRNA level in Acinetobacter bacteria, from the viewpoint of enabling more efficient translocation into the periplasm, a signal peptide derived from the Omp38 protein is preferred. The "signal peptide derived from the Omp38 protein" is typically a peptide containing the amino acid sequence set forth in SEQ ID NO: 14. However, as long as translocation into the periplasm is possible, it is not limited to the typical amino acid sequence, and its variants, mutants, and homologs can also be used. Examples of the variant or mutant of the signal peptide include a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 14. Here, "a plurality" is not particularly limited, but for example, it is within 10 amino acids (within 9 amino acids, within 8 amino acids, within 7 amino acids, within 6 amino acids, etc.), within several amino acids (within 5 amino acids, within 4 amino acids, within 3 amino acids, within 2 amino acids). Further, the amino acid modification is preferably a conservative substitution. In the present invention, "conservative substitution" means substitution with another amino acid residue having a similar (chemically similar side chain). Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention pertains. For example, among acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), and neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, valine, leucine, isoleucine, proline), amino acids having a hydroxy group (serine, threonine), amino acids containing sulfur (cysteine, methionine), amino acids having an amide group (asparagine, glutamine), amino acids having an imino group (proline), and amino acids having an aromatic group (phenylalanine, tyrosine, tryptophan) can be classified. Further, the amino acid sequence of the homolog of the signal peptide is an amino acid sequence having high homology (high similarity), preferably high identity, to the amino acid sequence set forth in SEQ ID NO: 14. High homology and the like are as described above.

[0079] In addition to the regulatory elements described above, the DNA construct of the present invention may have other sequences. Examples of such "other sequences" include marker genes for confirming the introduction into bacteria. For example, drug resistance genes, fluorescent protein genes, luminescent enzyme genes, and chromogenic enzyme genes can be mentioned. Specific examples of the "drug resistance gene" include kanamycin resistance gene, tetracycline resistance gene, gentamicin resistance gene, tellurite resistance gene, ampicillin resistance gene, etc. Specific examples of the "fluorescent protein gene" include GFP gene, mClover gene, mScarlet gene, DsRed gene, RFP gene, YFP gene, and aequorin gene. Specific examples of the "luminescent enzyme gene" include luciferase gene. Specific examples of the "chromogenic enzyme gene" include β-glucuronidase (GUS) gene, β-galactosidase gene, alkaline phosphatase gene, and SEAP gene.

[0080] <Method for producing protein> As shown in the examples described below, by culturing the above-mentioned bacteria into which a DNA construct capable of expressing the target protein has been introduced, it becomes possible to produce the protein substantially without endotoxin. Therefore, the present invention provides a method for producing a target protein, which includes a step of culturing the bacteria and a step of isolating the target protein from the culture obtained in the above step.

[0081] The method for introducing the DNA construct of the present invention into the bacteria of the present invention is not particularly limited, and examples thereof include electroporation method, rubidium chloride method, heat shock method, calcium chloride method, calcium phosphate method, lithium acetate method, conjugation transfer method, and method using calcium ions.

[0082] The method for culturing the bacterium of the present invention is not particularly limited, but it can be cultured using various media appropriately containing a carbon source, a nitrogen source, organic salts, inorganic salts, etc., which are used for normal culturing of bacteria belonging to the genus Acinetobacter. The culturing is carried out according to the normal method for bacteria belonging to the genus Acinetobacter, and it may be liquid culturing or solid culturing. However, as shown in the examples described later, according to the present invention, since it becomes possible to secrete and produce the target protein, liquid culturing is preferable. The culturing temperature and the pH of the medium can be appropriately selected from the range in which bacteria belonging to the genus Acinetobacter grow. For example, culturing conditions include culturing in a medium having a pH of about 6 to 8 at a culturing temperature of about 15 to 40 °C (usually 37 °C). The culturing time varies depending on various culturing conditions, but is usually from 1 day to 1 week. When an inducible promoter such as IPTG is used, the induction time is preferably within 1 day (usually 20 hours), but it may also be several hours.

[0083] The "cultured product" obtained by such culturing may be the proliferated bacterium of the present invention, may be something containing the bacterium (a culture solution containing the proliferated bacterium, a solid medium, etc.), or may be a medium (culture supernatant, etc.) from which the bacterium proliferated from these media has been removed.

[0084] The target protein can be purified from such a cultured product by a known protein purification method. For example, it can be purified by chromatography (ion exchange affinity chromatography, size exclusion column chromatography, affinity chromatography using an antibody that specifically recognizes the target protein, etc.), centrifugation, etc. Also, when the target protein contains the above-mentioned purification tag peptide, it can be purified by using a purification method corresponding to the tag peptide. For example, when using a histidine tag peptide, the target protein can be purified by binding it to a metal chelate resin, and when using a GST peptide, the target protein can be purified by binding it to a GST affinity resin.

[0085] <Kit> The present invention also provides a kit for producing a target protein, which at least contains the above-mentioned bacteria and DNA construct. In the kit of the present invention, in addition to these articles, a medium for culturing the bacteria of the present invention, a reagent for introducing the DNA construct of the present invention into bacteria, articles for purifying the target protein (chromatography, an antibody that specifically recognizes the target protein, a purification resin corresponding to a tag peptide, etc.) may also be included. Furthermore, it may include an instruction manual describing the method for producing the target protein according to the present invention.

Examples

[0086] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. Also, these examples were carried out using the methods and materials shown below.

[0087] <Methods and Materials> (Bacterial strains used) Escherichia coli BL21(DE3) for protein expression was purchased from New England BioLabs (Hitchin, UK), and Escherichia coli ClearColi with reduced endotoxin responsiveness was purchased from Lucigen (Middleton, Wisconsin, USA). Brevibacillus brevis for Gram-positive bacterial protein expression was purchased from Takara Bio Inc. (Shiga, Japan). Acinetobacter baumannii ATCC19606 strain was purchased from ATCC (Manassas, Virginia, USA).

[0088] (Establishment of endotoxin-free strains) The KL037S strain substantially free of endotoxin was isolated previously (see Non-Patent Document 18). Briefly, the culture broth of ATCC 19606 cultured in Luria-Bertani (LB) liquid medium was directly inoculated onto an LB agar medium (BD Biosciences, San Diego, California, USA) containing 10 μg / mL of colistin sulfate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and cultured at 37°C for 24 hours. From the colonies formed on the colistin-containing agar medium, LPS-deficient strains (KL037S strain, mutant type: 475_689(215)del:D159fs(D159C)D161*) having a mutation in the lpxC gene were obtained.

[0089] (Assay using horseshoe crab blood cell extract (LAL)) The LAL assay was performed according to the manufacturer's protocol using a Toxicolor LS-50M set (Seikagaku Corporation, Tokyo, Japan). To quantify the amount of endotoxin in bacteria, the overnight bacterial culture was washed with pyrogen-free physiological saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) and adjusted to an optical density (OD600) of 0.1 at 600 nm for measurement. The amount of endotoxin in the sample was calculated in units of EU / mg based on the amount of protein quantified by the BCA protein assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0090] (RNA sequencing analysis) Bacteria were inoculated into LB liquid medium and cultured with shaking at 37°C until the OD600 reached 0.7. Using NucleoSpin RNA (manufactured by Takara Bio Inc.), approximately 1×10 9Total RNA was extracted from CFU (colony forming unit) cells according to the manufacturer's protocol. DNase (Takara Bio Inc.) treatment was performed to completely remove genomic DNA, and rRNA was removed (using the NEBNext rRNA Removal Kit (New England BioLabs)). An RNA library was then prepared (using the MGIEasy Directional RNA Library Preparation Reagent Set (MGI Tech Co., Ltd., Wuhan, China)). Sequencing of the obtained library was outsourced to Genome-Lead (Kagawa, Japan), and 150-bp paired-end data were obtained using the DNBSEQ-G400RS sequencer (MGI Tech Co., Ltd.). The sequencing data were registered in the DNA Data Bank of Japan (DDBJ) (BioProject accession no. PRJDB16719). The acquired data were trimmed with Trim-Galore and mapped to ATCC 19606 (DDBJ nucleotide accession no. AP025740) using HISAT2 to obtain read count data. The count data were normalized with DESeq2.

[0091] (Preparation of Recombinant Protein Expression Plasmid) The plasmid pET26b(+), an Escherichia coli protein expression vector, was purchased from Merck Millipore (Burlington, Massachusetts, USA).

[0092] The plasmid pTAKE, a shuttle vector for recombinant protein expression in Escherichia coli - Acinetobacter, was constructed as follows. First, an IPTG-inducible protein expression cassette consisting of the lacI-tac promoter region, N-terminal 7× histidine tag, FactorXa protease cleavage site, multiple cloning site (MCS), and rrnB gene terminator of Escherichia coli K-12 MG1655 strain in pMAL-c2e (New England Biolabs Inc., MA, USA) was inserted into the Sse8387I-EcoRI site of pHSG298 (Takara Bio Inc.). The pWH1266 ori region of the Escherichia coli - Acinetobacter shuttle vector pKAMO was amplified by PCR and incorporated into the plasmid by In-Fusion HD cloning (Takara Bio Inc.) to construct the pTAKE vector (for pKAMO, see Non-Patent Document 19). The sequences of the PCR primers and template used for the construction of pTAKE are shown in Table 3 below. The schematic of pTAKE is shown in Figure 1.

[0093]

Table 3

[0094] Plasmid pTakeNO was constructed by inserting the signal peptide of Omp38 from A. baumannii (KAMO5_06030, signal peptide sequence: KLSRIALATMLVAAPLAAANAGV, SEQ ID NO: 14) into the 5' end of the target protein coding sequence of the pTAKE vector. Single-stranded oligonucleotides CTAGa-omp38-signalP-A_F (5'-CTAGAATGAAATTGAGTCGTATTGCACTTGCTACTATGCTTGTTGCTGCTCCATTAGCTGCTGCTAATGCTGGCGTAA-3', SEQ ID NO: 25) and omp38-signalP-ACTAG_R (5'-CTAGTTACGCCAGCATTAGCAGCTAATGGAGCAGCAACAAGCATAGTAGCAAGTGCAATACGACTCAATTTCATT-3', SEQ ID NO: 26) were annealed to prepare double-stranded DNA encoding the Omp38 signal peptide. pTAKE was cleaved with the restriction enzyme SpeI (Takara Bio Inc.), and ligated with the double-stranded DNA of the Omp38 signal peptide using a DNA ligation kit <Mighty Mix> (Takara Bio Inc.). A sequence encoding a 7× histidine tag was inserted into the C-terminus of the target protein coding sequence of the pTakeNO plasmid to construct pTakeCO.

[0095] The gfp and mouse tnfa genes were inserted into pTAKE using In-Fusion HD (Takara Bio Inc.). Genes encoding mNb6-tri-20aa (SARS-Cov-2 nanobody) and ATN-103 (ozoralizumab) were artificially synthesized (Eurofins Genomics, Tokyo, Japan) and inserted into pTakeCO and pTakeNO, respectively. The sequences of the PCR primers and templates used for the construction of these expression plasmids are shown in Table 4 below.

[0096]

Table 4

[0097] These plasmids were transformed into KL037S, ClearColi, and BL21(DE3) by electroporation. These bacteria were selected on LB agar medium containing 20 μg / ml kanamycin and used as protein expression strains.

[0098] (Expression Protein Purification) The strain harboring the expression vector of the target protein was cultured with shaking at 37 °C and 180 rpm for 20 h in LB liquid medium containing 20 μg / ml kanamycin. The cells cultured overnight were diluted 10-fold with LB liquid medium and cultured with shaking (180 rpm) until the OD600 reached 0.7 - 1.0. IPTG (FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 1 mM, and the cells were cultured with shaking at 37 °C for 20 h (135 rpm). After centrifugation (13000 g, 15 min, 4 °C), the supernatant was collected. The cells obtained from 2 ml-scale and 100 ml-scale cultures were washed with PBS and resuspended in 300 μl and 10 ml of xTractor Buffer (Takara Bio Inc.), respectively, and incubated with rotation mixing at 4 °C for 15 min. After centrifugation (13000 g, 20 min, 4 °C), the supernatant was collected as the cell lysate. The expressed protein was purified from the supernatant and cell lysate using Ni-NTA agarose (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.).

[0099] Purification from 2 ml of the culture was performed by the batch method using 40 μl of Ni-NTA agarose (50% suspension). Ni-NTA agarose was mixed with the culture supernatant or cell lysate and rotated and mixed at 4 °C for 1 h. The Ni-NTA agarose bound with the protein was washed twice with 1 ml of buffer containing 10 mM imidazole and eluted with 100 μl of buffer containing 250 mM imidazole. 10 μl of the eluate was separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) under reducing conditions, and the protein was visualized by CBB (Coomassie Brilliant Blue) staining.

[0100] Purification from 100 ml of the culture solution was performed by a column method using 1 ml of Ni-NTA agarose (50% suspension). The Ni-NTA agarose bound with the protein was washed with 20 ml of a buffer containing 10 mM imidazole and eluted with 1.5 ml of a buffer containing 250 mM imidazole. Using Amicon Ultra (Merck Millipore), the buffer of the eluate was replaced with pyrogen-free physiological saline and concentrated. For the concentration of GFP or mouse TNF-α, Amicon Ultra with a molecular weight cut-off of 3K was used. For the concentration of mNb6-tri-20aa or ATN-103, Amicon Ultra with a molecular weight cut-off of 30K was used.

[0101] The protein concentration was quantified using a BCA protein assay according to the manufacturer's protocol. 1 μg of the protein solution was separated by SDS-PAGE under reducing conditions and visualized by CBB staining.

[0102] (Measurement of the activity of tumor necrosis factor (TNF)-α) The TNF-sensitive mouse fibroblast cell line L929 (RCB2619) was purchased from RIKEN BRC (Tsukuba, Japan). The measurement of TNF-α activity was performed with some modifications to the method described on the RIKEN BRC homepage (https: / / cell.brc.riken.jp / ja / rcb / bioassay_l929). One day before the addition of mouse TNF-α, 1.5×10 4 cells were seeded in each well of a 96-well plate. The concentration of purified mouse TNF-α was measured by ELISA (mouse TNF-alpha DuoSet ELISA, R&D Systems, Minneapolis, MN, USA). After washing the cells with RPMI1640 medium (FUJIFILM Wako Pure Chemical Industries), serial three-fold dilutions of mouse TNF-α and 4 μg / ml of actinomycin D (FUJIFILM Wako Pure Chemical Industries) were added, and the cells were cultured at 37 °C under a 5% CO2 partial pressure for 18 hours. Cell viability was measured using an MTT assay (Dojindo Laboratories, Kumamoto, Japan).

[0103] (Measurement of the SARS-Cov-2 virus neutralizing activity) One day before virus infection, 10 4 VeroE6 cells were seeded in each well of a 96-well plate. Serial 10-fold dilutions of mNb6-tri-20aa (SARS-Cov-2 nanobody) or ATN-103 as a negative control nanobody were prepared in Opti-MEM medium (Thermo Fisher Scientific), and then wild-type (Wuhan) or Delta strain of coronavirus was added and incubated at 37 °C for 1 hour. Then, the virus-nanobody solution was added to the plate on which VeroE6 cells were cultured at an MOI (multiplicity of infection) of 0.01. Three days after culturing, an MTT assay (Dojindo Laboratories, Kumamoto, Japan) was performed to measure virus activity from cell viability.

[0104] (Enzyme-linked immunosorbent assay (ELISA)) For the evaluation of expressed and purified ATN-103 (ozoralizumab; anti-human TNF-α nanobody), Human TNF-α DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) was used. The capture antibody was changed to 4 μg / mL of ATN-103, and basically the manufacturer's protocol was followed. mNb6-tri-20aa was used as a negative control nanobody, and the standard product of human IL-8 (Human IL-8 / CXCL8 DuoSet ELISA (R&D Systems)) was used as a negative control for cytokines, respectively.

[0105] (Growth experiment) The growth ability was adjusted so that the OD600 of the bacteria cultured overnight was 0.001, and static culture was performed at 37 °C for 24 hours in LB medium. Using Multiskan FC (Thermo Fisher Scientific), the absorbance at 595 nm (A595nm) was measured every hour.

[0106] (Cloning of various VHHs) The amino acid sequences of various VHHs were optimized for the codons of A. baumnnii with reference to the literature and other sources, and then artificially synthesized (Twist Bioscience). Specifically, for GFP VHH, refer to Kato Y et al., J Cell Sci. 2015 Jun 15;128(12):2351-62.doi:10.1242 / jcs.168740.Epub 2015 May 11.PMID:25964651.; for mouse TNF-α VHH (VHH#m3F) and mouse albumin VHH (MSA21), refer to the patent publication of ozoralizumab (ATN-103) (WO2004 / 041862A2); for mouse MMP-8 VHH, refer to Delphine Demeestere et al., Mol Ther. 2016 May;24(5):890-902.doi:10.1038 / mt.2016.2.Epub 2016 Jan 18.PMID:26775809. For mouse CD11b VHH and mouse MHC class II VHH, refer to Mohammad Rashidian et al., Proc Natl Acad Sci USA. 2015 May 12;112(19):6146-51.doi:10.1073 / pnas.1502609112.Epub 2015 Apr 20.PMID:25902531, and DNA encoding various VHHs was prepared. Then, each gene was inserted into pTakeCO or pTakeNO to construct expression plasmids.

[0107] (Western blotting) The purified GFP VHH and mTNF-α VHH were adjusted to 1 mg / mL using PBS, mixed with 1 mM Biotin-NHS (Cayman Chemical), and reacted at room temperature for 30 minutes. Then, unreacted Biotin-NHS was removed, washed, and concentrated using a 3K Amicon Ultra. Protein concentration was quantified using a BCA protein assay kit.

[0108] A 1 μg protein solution of GFP, mTNF-α, or BSA (bovine serum albumin, Sigma) was separated by SDS-PAGE under reducing conditions and visualized by CBB staining.

[0109] Similarly, the 1 μg protein solution was separated by SDS-PAGE under reducing conditions and transferred to a PVDF membrane (Merck Millipore) using a semi-dry transfer apparatus (BioCraft). Subsequently, blocking was performed with EzBlock Chemi (ATTO), and biotinylated VHH (1 μg / mL) was used as the primary antibody and reacted overnight at 4°C. Then, detection was performed using streptavidin-HRP (Cell Signaling Techenology) as the secondary antibody.

[0110] (ELISA using GFP VHH and mTNF-α VHH) GFP, mTNF-α, or BSA was added to a 96-well plate at a concentration of 4 μg / mL and subjected to a coating treatment overnight at 4°C. The wells were washed three times with PBS containing 0.05% Tween20 and once with PBS, and then blocking treatment was performed with PBS containing 1% BSA at room temperature for 1 hour. Subsequently, biotinylated VHH at various concentrations (10, 50, 100, 500 ng / mL) was reacted at room temperature for 2 hours. Then, the wells were washed in the same manner as above, streptavidin-HRP (attached to Human TNF-α DuoSet ELISA) was added, reacted at room temperature for 20 minutes, and the reaction was stopped by adding H2SO4.

[0111] (Preparation of artificially gene-modified strains) For homologous recombination, the pSBKT5v2 (drug resistance gene: Kanamycin resistance gene, counter-selection gene: tdk) plasmid was used. Approximately 1.5 kbp before and after each target gene of A.baumannii was subcloned into pSBKT5v2 using In-Fusion reagent (TaKaRa, Shiga, Japan). The prepared plasmid was transformed into S17-1 λpir by heat shock.

[0112] The gene mutant strain was prepared using the double homologous recombination method. S17-1 λpir carrying plasmid pSBKT5v2 and A. baumannii were conjugally transferred on LB agar for 3 - 6 hours. Then, the bacterial cells were collected and A. baumannii was selected on M9 agar (Sigma - Aldrich, St. Louis, USA) with 0.3% citric acid (Fuji Film Wako Pure Chemical Industries) as the carbon source and M9 agar containing 50 μg / mL Kanamycin (Fuji Film Wako Pure Chemical Industries). The formed colonies were subjected to blue - white selection on X - gal agar (Eiken Chemical, Tokyo, Japan) containing 50 μg / mL Kanamycin to recover only A. baumannii. The pSBKT5v2 - introduced strain induces tdk for counter - selection by IPTG. A. baumannii in which single recombination was completed using pSBKT5v2 was inoculated into LB broth containing 50 μg / mL Kanamycin and cultured at 37°C and 135 rpm for 18 - 20 hours. Then, it was sub - cultured in LB broth without antibacterial agents. When the OD600 reached 0.5 - 0.8, IPTG was added to a final concentration of 1 mM and cultured for 3 hours. Counter - selection was performed on LB agar coated with 100 μL of 200 μg / mL Azidothymidine with the bacterial solution.

[0113] The results obtained using the above materials and methods are shown below.

[0114] <Example 1> Examination of endotoxin - free bacterial strains To construct an expression system for a recombinant protein substantially free of endotoxin (LPS), the bacteria used for it were evaluated using the LAL assay. The evaluation targets were Acinetobacter baumannii KL037S strain (see Non - Patent Document 18) showing resistance to colistin, which the inventors had previously found, ClearColi, and Brevibacillus, which have already been used in an expression system with reduced endotoxin contamination.

[0115] The LAL assay is the gold standard for detecting endotoxin contamination. In the presence of endotoxin, LAL coagulates, enabling highly sensitive detection of endotoxin. ClearColi has its native hexaacyl lipid A of LPS converted to tetraacyl lipid A (lipid IVA) to suppress the endotoxin reaction. However, since the coagulation reaction of the LAL assay is induced by the 4'-monophosphoryl diglucosamine skeleton of LPS, lipid IVA having such a skeleton, i.e., ClearColi, also shows a positive result in the evaluation by the said assay. Further, in the LAL assay, the coagulation of LAL is induced not only by endotoxin derived from Gram-negative bacteria but also by fungal β-glucan and lipoteichoic acid (LTA) of Gram-positive bacteria. Therefore, Brevibacillus, which is a Gram-positive bacterium, also shows a positive result in the evaluation by the said assay.

[0116] As a result of the evaluation by such an assay, as shown in Fig. 2, the endotoxin concentration of the bacterial suspension (OD600: 0.1) of the KL037S strain was 0.030 ± 0.002 EU / mL. On the other hand, the endotoxin concentration of the ClearColi strain of E. coli commercially available as a host for recombinant protein expression with reduced endotoxin activity was 2.1×10 3 ± 0.33×10 3 EU / mL. That is, it became clear that the endotoxin concentration of the KL037S strain was 1 / 70000 of that of ClearColi. Also, even in Brevibacillus, a Gram-positive bacterium used as a host for recombinant protein expression, although it has no LPS, the endotoxin level was 1.6 ± 0.15 EU / mL, which was found to be about 53 times higher than that of the KL037S strain.

[0117] Therefore, using this KL037S strain, an attempt was made to develop an endotoxin-free recombinant protein expression system as shown below.

[0118] <Example 2> Production of GFP We attempted to produce GFP, which is the gold standard of recombinant proteins, using the endotoxin-free strain KL037S. Specifically, for recombinant protein expression, an E. coli-Acinetobacter shuttle vector (pTAKE) was constructed (Figure 1). The pTAKE vector contains a lac operator after the tac promoter, and the expression of downstream genes is induced in the presence of IPTG (isopropyl-β-D-thiogalactopyranoside).

[0119] As a result of culturing the KL037S strain into which the pTAKE vector encoding GFP was introduced, we successfully purified GFP from the cell lysate on a 2 mL culture scale (Figure 3A). We also succeeded in purification from a 100 mL culture scale (Figure 3B). Furthermore, it was confirmed that the GFP purified from the KL037S strain is a functional protein that emits fluorescence, similar to the GFP purified from ClearColi (Figure 3C). The yield of GFP was 0.38 ± 0.16 mg / 100 mL in the KL037S strain and 1.7 ± 0.25 mg / 100 mL in ClearColi. On the other hand, the endotoxin level in the purified GFP fraction was 0.14 ± 0.01 EU / mg in the KL037S strain, while it was 117 ± 109 EU / mg in the ClearColi strain. It became clear that the amount of endotoxin was significantly reduced to approximately 1 / 836 in recombinant protein production in the KL037S strain compared to ClearColi (Figure 3D). That is, it became clear that by using the endotoxin-free KL037S strain as a host, recombinant proteins can be produced at a significantly low endotoxin level.

[0120] <Example 3> Production of Cytokine In the field of biopharmaceuticals and the like, it is important to produce cytokines endotoxin-free. Therefore, we attempted to produce mouse TNF-α using the above endotoxin-free strain (KL037S strain). Specifically, a pTAKE vector inserted with the mouse-derived tnfa gene was introduced into the KL037S strain and cultured, and an attempt was made to detect TNF-α in the obtained cell lysate. As a result, IPTG-inducible production of TNF-α was observed (Figure 4A).

[0121] Interestingly, TNF-α was also secreted into the culture supernatant, and the contamination of more non-specific proteins was lower than that in the cell lysate. Therefore, as a result of purifying TNF-α from 100 mL of the culture supernatant, the yield and endotoxin level at that time were 0.12 ± 0.04 mg / 100 mL and 3.6 ± 2.0 EU / mg, respectively (Figure 4B). Furthermore, the activity of the purified TNF-α was evaluated by a cell viability assay using TNF-sensitive mouse fibroblasts. As a result, the cell survival rate decreased in a concentration-dependent manner, and the EC 50 was 24 pg / mL (Figure 4C).

[0122] On the other hand, the endotoxin level and EC 50 of the positive control (recombinant TNF-α (#T7539) manufactured by Sigma-Aldrich) for the activity evaluation of TNF-α published on the RIKEN BRC website (https: / / cell.brc.riken.jp / ja / rcb / bioassay_l929) were <1000 EU / mg and 10 - 500 pg / mL, respectively. Also, the endotoxin levels of other commercially available recombinant TNF-α are usually about <100 - 1000 EU / mg.

[0123] Therefore, it has been revealed that, compared with currently commercially available recombinant protein products, the recombinant protein expression system using this endotoxin-free strain as a host can produce functional TNF-α with extremely low levels of endotoxin contamination. Although not shown in the figures, it has also been confirmed that mouse interferon (IFN)-γ can be prepared from the cell lysate of this endotoxin-free strain at extremely low levels of endotoxin.

[0124] <Example 4> Production of Nanobodies In recent years, the development of the nanobody field has been remarkable. Therefore, considering the application of the endotoxin-free recombinant protein expression system to this field, in order to demonstrate the usefulness of the system, we attempted to produce two types of nanobodies. One is mNb6-tri-20aa, a multivalent antibody in which three nanobodies are bound to improve virus neutralizing activity, and this antibody specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The other is ozoralizumab (ATN-103), a multispecific antibody combining two anti-human TNF-α VHHs and one anti-human albumin VHH, which is a pharmaceutical approved for use in rheumatoid arthritis patients in Japan in September 2022. However, for both of these two antibodies, no purification from the culture supernatant and cell lysate was observed even when using the pTAKE and KL037S strains (Figure 5A).

[0125] Therefore, as a result of intensive studies to enable further production of low-molecular antibodies and the like, we came up with the idea of using a periplasmic translocation signal sequence. First, in order to find a signal sequence suitable for the genus Acinetobacter, RNA sequence analysis of the KL037S strain and its parental strain ATCC19606 was performed. As a result, high expression of the mRNA encoding the outer membrane protein omp38 (KAMO5_06030) was observed in both strains (Figure 5B). Next, a plasmid (pTakeNO / CO) was constructed to add the signal peptide sequence of Omp38 to the N-terminus of the recombinant protein for expression (Figure 5C). DNA encoding the above low-molecular antibodies was inserted into the plasmid (pTakeCO-mNb6-tri-20aa or pTakeNO-ATN-103) and introduced into the KL037S strain. Then, attempts were made to detect each low-molecular antibody in the cultures of these transformants. As a result, these antibodies could be produced in an IPTG-inducible manner, and purification from the culture supernatant was successful (Figure 5D).

[0126] Next, the endotoxin levels and functions of each low-molecular antibody purified from the culture supernatant were evaluated. As a result, the yield of mNb6-tri-20aa was 0.13 ± 0.01 mg per 100 mL of culture supernatant, and the endotoxin level was 0.26 ± 0.06 EU / mg. The yield of ATN-103 was 0.11 ± 0.02 mg / 100 mL, and the endotoxin level was 0.99 ± 0.50 EU / mg. Also, both low-molecular antibodies were purified in a pure form as the expected trimer, and monomers and dimers were also slightly observed (Figure 6A).

[0127] Next, the activity of the anti-SARS-CoV-2 antibody mNb6-tri-20aa was evaluated by a coronavirus neutralization assay. As a result, virus activity neutralization against both the SARS-CoV-2 wild strain (Wuhan strain) and the Delta variant was observed in a concentration-dependent manner (Figure 6B). Also, the IC 50It was 820 pM and 1.9 nM for the wild strain (Wuhan strain) and the Delta variant, respectively. On the other hand, the IC 50 of mNb6-tri-20aa derived from BL21(DE3) cell lysate was 1.9 nM for both strains. Also, ATN-103 did not show neutralizing activity against SARS-CoV-2 even at high concentrations.

[0128] Initially, the combination of ClearColi and pET26b(+) was used to attempt the production of the two small antibodies on a 2 mL culture scale, but it was found that the control by IPTG was not effective. Also, when scaling up to 100 mL culture, poor bacterial growth occurred due to toxicity and the purification of the target antibody was not achieved. Therefore, mNb6-tri-20aa was expressed using the combination of BL21(DE3) strain and pET26b(+), and the purified product from its cell lysate was used as the comparison object of the present invention as described above. Also, as an alternative means, this combination was used to attempt the purification of mNb6-tri-20aa on a 100 mL culture scale, and although the purification was successful, a lot of non-specific protein contamination and a high endotoxin level were confirmed (endotoxin level: 5.9×10 4 ±4.6×10 4 EU / mg, yield: 0.22±0.07 mg / 100 mL).

[0129] Next, the activity of Ozoralizumab (ATN-103) was evaluated by ELISA. Specifically, the capture antibody of the human TNF-α ELISA kit was replaced with ATN-103 and the assay was performed. As a result, ATN-103 showed a concentration-dependent response when detecting a dilution series of TNF-α, and the correlation coefficient was R 2 =0.9979 (Figure 6C). On the other hand, for the negative control cytokine (interleukin-8 (IL-8)), it was not detected at all by ATN-103. Furthermore, mNb6-tri-20aa also did not recognize TNF-α at all (Figure 6D).

[0130] From the above results, it became clear that by using the above signal peptides in combination, the extracellular secretion production of recombinant proteins such as low molecular antibodies can be carried out more efficiently. That is, it also became clear that the purification of such recombinant proteins from the culture supernatant can be carried out more simply with minimal endotoxin contamination without requiring cell lysis, disruption, or other complex procedures. As a result, cost reduction in the purification of recombinant proteins and reuse of the bacterial cells can be expected. Furthermore, it was confirmed that the recombinant proteins obtained in this way maintain their functions.

[0131] <Example 5> Improvement of Endotoxin-Free Strain In the production of recombinant proteins, in addition to the contamination of the above endotoxin, the contamination of host-derived proteins also becomes a problem. In this regard, even in the system using the above endotoxin-free strain (KL037S strain), when purifying the protein that binds to nickel sepharose from the culture supernatant of the bacterial strain without the recombinant protein expression plasmid, purification of a protein of about 12 kDa was observed regardless of the addition of IPTG (Figure 7). That is, it means that non-specific proteins are mixed into the recombinant proteins purified from the culture supernatant in this system. Actually, in ATN-103 purified from the culture supernatant of KL037S strain, non-specific bands were confirmed at the front line of electrophoresis (the right photograph in Figure 5D). Note that since the ATN-103 used for functional evaluation was concentrated and solvent-exchanged with a 3K Amicon, this contamination was not observed.

[0132] However, on the other hand, no non-specific bands were detected in mTNF-α and mNb6-tri-20aa similarly purified from the culture supernatant of KL037S strain (the left photograph in Figure 4A and Figure 5D). Thus, since a strain in which non-specific bands were not detected was confirmed in the transformant of KL037S strain, it was shown that in some strains, the production of non-specific proteins may have been suppressed during the plasmid introduction process.

[0133] Therefore, expecting the reproduction of transformants without non-specific bands detected, the KL037S strain was subjected to electroporation without plasmid addition. Subsequently, the diluted bacterial solution was seeded on an LB agar medium (without drugs), and Eep2 was established from the formed colonies (Eep: empty electroporation). As a result of purifying the protein that binds to nickel sepharose from the culture supernatant of the Eep2 strain, unlike the parental strain (KL037S strain), a protein of about 12 kDa was not purified (Figure 7).

[0134] Furthermore, as a result of evaluating the endotoxin concentration of the bacterial suspension (OD600: 0.1) of the Eep2 strain using the LAL assay, it was 0.015 ± 0.005 EU / mL. This value was comparable to the endotoxin concentration of the KL037S strain (0.048 ± 0.007 EU / mL), and it was confirmed that the Eep2 strain was also an endotoxin-free strain.

[0135] In addition, for the KL037S strain, in addition to the above-mentioned contamination of non-proteins, there is a problem that its growth ability is lower compared to its parental strain (Figure 8). Therefore, when the growth ability of the Eep2 strain was evaluated, an improvement in the growth ability from the KL037S strain was observed (Figure 8).

[0136] <Example 6> Production of small molecule antibodies in recombinant protein expression using the Eep2 strain To evaluate the usefulness of the Eep2 strain, attempts were made to produce nanobodies against GFP or mouse TNF-α. As a result, by culturing the Eep2 strain introduced with pTakeCO-GFP VHH or pTakeNO-mTNF-α VHH, GFP VHH or mTNF-α VHH was successfully purified from these culture supernatants in an IPTG-inducible manner (Figure 9A).

[0137] In addition, when culturing was performed on a 100 mL scale and attempts were made to purify each nanobody from these culture supernatants, the yield of GFP VHH was 0.45 ± 0.11 mg / 100 mL, and the yield of mTNF-α VHH was 0.07 ± 0.02 mg / 100 mL.

[0138] Next, the functions of each purified nanobody were evaluated. Specifically, a biotin-labeled nanobody was used as the primary antibody for Western blotting. As a result, it was confirmed that each nanobody could specifically recognize each protein (Figure 9B). Furthermore, in order to evaluate the specificity for non-denatured proteins, ELISA was also performed. As a result, it was confirmed that the nanobody could specifically recognize each protein even by ELISA (Figure 9C).

[0139] From the above results, it became clear that the VHH obtained using the Eep2 strain could specifically recognize the antigen protein regardless of denaturation or non-denaturation. That is, according to the recombinant protein expression system using the Eep2 strain, it became clear that the protein could be produced while maintaining its function.

[0140] Next, in the same manner as in <Example 4> above, attempts were made to produce other small antibodies using the Eep2 strain. As a result, for all of Ozoralizumab (ATN-103), mouse Albmin VHH, mouse MMP (matrix metalloproteinase)-8 VHH, mouse CD11b VHH, and mouse MHC (major histocompatibility complex) class II VHH, successful purification from the culture supernatant was induced by IPTG (Figures 10A to 10C).

[0141] <Example 7> Gene analysis of the Eep2 strain As described above, the Eep2 strain is superior to its parental strain (KL037S strain) in terms of suppressing non-specific protein expression and growth ability. Therefore, in order to identify the responsible genes for these phenotypes, mutation analysis was performed by NGS. As a result, as shown in Table 5 below, a single-base deletion mutation was detected in each of the baeR gene (Locus_tag: KAMO5_05630) and the polyhydroxyalkanoate granule-binding protein (Phasin) family protein gene (Locus_tag: KAMO5_18120) in the Eep2 strain. Furthermore, an insertion mutation of ISAba11 (transposon) was found in the phosphatidic acid phosphatase type 2 (PAP2) family protein gene (Locus_tag: KAMO5_07110) in the Eep2 strain.

[0142]

Table 5

[0143] These mutations are thought to cause frameshifts and disrupt / decrease the functions of the proteins encoded by the respective genes. Furthermore, regarding the above three genes, when the mRNA expression levels in the KL037S strain were evaluated by RNA sequence analysis, the baeR gene was 2^3.19-fold, the Phasin family protein gene was 2^-1.79-fold, and the PAP2 family protein gene was 2^1.54-fold compared to the parental strain ATCC19606.

[0144] In addition, BaeR is known to be a molecule that functions as a two-component regulatory system together with BaeS. Therefore, the mRNA level of the baeS gene was also evaluated. As a result, in the KL037S strain, it increased 2^3.63-fold compared to the parental strain ATCC19606.

[0145] Furthermore, in view of the above results, Sanger sequence analysis of the baeRS gene was performed on the KL037S strain (pTAKE-mTNF-α in KL037S, pTakeCO-mNB6-tri-20aa in KL037S) into which pTAKE-mTNF-α or pTakeCO-mNb6-tri-20aa, in which no non-specific band was detected in the culture supernatant, was introduced. As a result, in both strains, a 173T>A (amino acid substitution L58Q) mutation was detected in the baeS gene. On the other hand, no mutation was observed in the baeRS gene in the KL037S strain into which pTakeCO-ATN-103, in which a non-specific band was detected in the culture supernatant, was introduced.

[0146] Therefore, it was clarified that the disruption / decrease of the function of BaeRS suppresses the expression of proteins in the culture supernatant that non-specifically bind to nickel sepharose. Next, in order to attempt to improve the decrease in growth associated with LPS deficiency, first, an artificial LPS-deficient strain was prepared by disrupting the lpxA gene, which acts at the initial stage of LPS biosynthesis. Then, based on the LPS-deficient strain, a combined artificial-deficient strain of three genes (baeR, PAP2 (KAMO5_07110), phasin (KAMO5_18120)) in which disruption was observed with Eep2 was prepared, and the growth ability was evaluated. As a result, the growth ability, which was significantly decreased by lpxA deficiency, was partially improved by adding baeR gene deficiency, and a remarkable improvement in growth ability was observed by further adding PAP2 gene deficiency (Figure 11). As a result, the lpxA-baeR-PAP2 gene-deficient strain showed a growth ability comparable to that of the Eep2 strain, and it became clear that the responsible genes for the improvement in growth ability in the LPS-deficient strain observed in the Eep2 strain were the two genes, baeR and PAP2. From the above results, the strain in which the lpxA+baeR+PAP2 genes were disrupted is the optimal strain as a recombinant protein expression host that suppresses non-specific protein production and also restores the growth ability.

Industrial Applicability

[0147] As described above, according to the present invention, it is possible to produce a target protein substantially without endotoxin. In particular, according to the present invention, it is possible to produce a target protein with almost complete elimination of endotoxin contamination, such that it is negative in the LAL assay, which is the most sensitive and reliable method for detecting bacterial endotoxin. In addition, it is also possible to efficiently grow the bacterium serving as the expression host of the target protein, and further suppress the expression and contamination of non-specific proteins while producing the protein. Further, according to the present invention, it is also possible to more efficiently perform extracellular secretion production while maintaining the function of the target protein.

[0148] Therefore, the protein production method of the present invention is extremely useful in fields such as the production of biopharmaceuticals such as cytokines and antibodies.

Claims

1. A bacterium belonging to the genus Acinetobacter in which the functions of a lipopolysaccharide (LPS) biosynthetic protein and a BaerR protein and / or a Baes protein are suppressed.

2. The bacterium according to claim 1, wherein the function of a phosphatidic acid phosphatase type 2 (PAP2) family protein is further suppressed.

3. The bacterium according to claim 1, wherein the LPS biosynthetic protein is at least one protein selected from the group consisting of an LpxC protein, an LpxA protein, and an LpxD protein.

4. The bacterium according to claim 1, into which a DNA construct capable of expressing a target protein has been introduced.

5. The bacterium according to claim 4, wherein the DNA construct is a DNA construct capable of expressing a target protein by fusing a periplasm translocation signal peptide to the N-terminus of the target protein.

6. The bacterium according to claim 5, wherein the periplasm translocation signal peptide is a signal peptide derived from an Omp38 protein.

7. A method for producing a target protein, comprising: culturing the bacterium according to any one of claims 4 to 6; and isolating the target protein from the culture obtained in the above step. A method comprising the above steps.

8. A kit for producing a target protein, comprising: the bacterium according to any one of claims 1 to 3; and a DNA construct capable of expressing a target protein.

9. The kit according to claim 8, wherein the DNA construct is a DNA construct capable of expressing a target protein by fusing a periplasm translocation signal peptide to the N-terminus of the target protein.

10. The kit according to claim 9, wherein the periplasm translocation signal peptide is a signal peptide derived from an Omp38 protein.

11. A DNA construct capable of expressing a target protein by fusing a signal peptide derived from an Omp38 protein to the N-terminus of the target protein.

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