Method for producing foreign proteins using Escherichia coli

Abstract

The present invention makes it possible to efficiently extract and purify a target foreign protein expressed using Escherichia coli from the periplasm.　A protein production method that includes steps (a)–(c).　(a) A step for introducing a gene that codes for a target foreign protein into Escherichia coli in which a gene associated with maintenance of outer membrane structure has been modified. (b) A step for culturing the Escherichia coli from (a). (c) A step for collecting the target foreign protein from the periplasmic fraction of the cultured bacterial cells.

Description

Technical Field

[0001] The present invention relates to a method for producing a foreign protein using Escherichia coli.

Background Art

[0002] In the production of recombinant proteins using gene recombination technology, various hosts are used. For example, animal cells such as CHO (Chinese Hamster Ovary) cells, and microorganisms such as Escherichia coli and yeast can be mentioned. Among microorganisms, there is an advantage that the target protein can be produced in a shorter time and at a lower cost compared to animal cells. In particular, Escherichia coli is one of the most studied microorganisms, and it is frequently used in protein production because it grows in a short time and is easy to handle.

[0003] In protein production using Escherichia coli, generally, the protein is produced intracellularly. Therefore, after fermentation culture, it is necessary to separate the culture solution into cells and culture supernatant, and perform operations such as periplasm extraction, cell disruption, or recovery of inclusion bodies. In particular, when cell disruption is involved, since it is necessary to purify the target protein from a variety of host-derived substances, these post-treatment steps greatly affect the production cost of the target protein.

[0004] Regarding foreign proteins, if a method for selectively releasing the contents of the periplasm without releasing the contents of the cytoplasm is used, contamination of host cell-derived proteins can be reduced. Therefore, the purification process of foreign proteins is easier compared to the purification process from the cytoplasm. In addition, since the periplasm is an oxidative environment, it is also known that by expressing a foreign protein in the periplasm, formation of intramolecular and intermolecular disulfide bonds and accompanying functional expression of the foreign protein are easily realized. Due to the above circumstances, in protein production, the technique of expressing a foreign protein in the periplasm has been widely implemented (for example, Patent Document 1 and Non-Patent Document 1). [[ID=二十一]] [[ID=二十二]]

Prior Art Documents

[0005] [Patent Document 1] Japanese Patent Application Publication No. 08-242879 [Non-patent literature]

[0006] [Non-Patent Document 1] C. Schimek et al., Biotechnol Progress.36: e2999 (2020) [Overview of the project] [Problems that the invention aims to solve]

[0007] As described above, the method of expressing a target foreign protein in E. coli and extracting the foreign protein from the E. coli periplasm is widely used. However, it is known that the extraction efficiency of the target foreign protein varies depending on the molecular weight and expression level of the foreign protein being expressed. Therefore, a method for efficiently obtaining foreign proteins from E. coli, regardless of their molecular weight, is desired.

[0008] The present invention aims to provide a method for producing proteins using E. coli, which enables efficient extraction and purification of a target foreign protein after localization of the E. coli periplasm. [Means for solving the problem]

[0009] As a result of diligent research, the inventors of this invention discovered that by using an Escherichia coli strain with modified genes related to the maintenance of the outer membrane structure, the extraction efficiency of expressed foreign proteins localized in the periplasm is increased, leading to the completion of this invention.

[0010] In other words, the following inventions are provided according to this specification. (1) A method for producing protein, comprising the following steps (a) to (c): (a) A step of introducing a gene encoding a target foreign protein into Escherichia coli in which genes related to the maintenance of the outer membrane structure have been modified. (b) The step of culturing E. coli as in (a), and (c) A step to recover the target foreign protein from the periplasmic fraction of the bacterial cells after culture. (2) The method according to (1), wherein the gene related to the maintenance of the outer membrane structure is at least one selected from the group consisting of pal, lpp, ompA, tolA, mepS, nlpI, arcA, bamD, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB. (3) The method according to (1) or (2), wherein two or more genes selected from the group consisting of pal, lpp, ompA, tolA, mepS, nlpI, arcA, bamD, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB of the Escherichia coli are modified. (4) The method according to (1) or (2), wherein at least one gene selected from the group consisting of pal, lpp, ompA and tolA of the Escherichia coli is modified. (5) The method according to any of (1) to (4), wherein the modification is completely absent. (6) The method according to any one of (1) to (4), wherein the modification is a partial mutation. (7) The method of (6) wherein the modification is a partial mutation of the signal sequence. (8) The method according to (6), wherein the modification is a partial mutation of a structural gene. (9) The method according to any one of (1) to (8), wherein the Escherichia coli is derived from strain B or strain K12. (10) A method for preparing a host Escherichia coli for the production of a protein, comprising the steps (i) and (ii) below: (i) the process of modifying genes related to the maintenance of the outer membrane structure of Escherichia coli; and (ii) The process of introducing a gene encoding the target foreign protein into the E. coli whose genes have been modified in (i). (11) The method according to (10), wherein the Escherichia coli expresses a target foreign protein, and the target foreign protein is accumulated in the periplasm of the Escherichia coli. This specification includes the disclosures of Japanese Patent Application No. 2021-046808, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a method for producing proteins using E. coli, which enables the efficient extraction and purification of a target foreign protein after localizing the foreign protein to the E. coli periplasm. [Modes for carrying out the invention]

[0012] 1. Method for producing proteins The present invention provides a method for producing protein, characterized by comprising the following steps (a) to (c). (a) A step of introducing a gene encoding a target foreign protein into Escherichia coli in which genes related to the maintenance of the outer membrane structure have been modified. (b) The step of culturing E. coli as in (a), and (c) A step to recover the target foreign protein from the periplasmic fraction of the bacterial cells after culture. The method of the present invention, having the above-mentioned features, is a method that can efficiently recover the target foreign protein expressed and localized in the periplasm of Escherichia coli.

[0013] In this specification, "foreign protein" means a protein encoded by a gene incorporated from outside the host cell, which is not normally expressed by a host cell that has not undergone transformation.

[0014] 1-1 Escherichia coli strain The Escherichia coli used in the method for producing the protein of the present invention (hereinafter also referred to as "the method of the present invention") is a genetically modified strain, but the Escherichia coli strain serving as its basis is not particularly limited, and any known Escherichia coli strain can be used. In particular, strain B or a strain derived from strain B, or strain K12 or a strain derived from strain K12 can be preferably used. Alternatively, the HB101 strain, which is a hybrid strain of strain B and strain K12, can be used. Examples of strains derived from strain B include the BL21 strain and the REL606 strain. Examples of strains derived from strain K12 include the W3110 strain, the DH10B strain, the BW25113 strain, the DH5α strain, the MG1655 strain, the JM109 strain, and the RV308 strain.

[0015] 1-2 Outer membrane formation-related genes The Escherichia coli strain used in the method of the present invention is a genetically modified strain in which one or more genes related to the maintenance of the outer membrane structure have been modified.

[0016] In the present specification, the "outer membrane formation-related gene" refers to a gene related to the maintenance of the outer membrane structure of Escherichia coli. More specifically, the "outer membrane formation-related gene" refers to a gene of Escherichia coli that is involved in the expression of a protein related to the maintenance of the outer membrane structure of Escherichia coli, that is, a nucleic acid (DNA or RNA, preferably DNA) possessed by Escherichia coli. The outer membrane formation-related gene is not particularly limited as long as it is a gene related to the maintenance of the outer membrane structure. For example, it is preferably the pal, lpp, ompA, tolA, mepS, nlpI, arcA, bamD, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB genes. In particular, it is more preferably the pal, lpp, ompA, or tolA gene, and even more preferably the pal or lpp gene. More specifically, it is preferably a gene having a nucleotide sequence represented by any of SEQ ID NOs: 4 to 32 and having a sequence identity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0017] In this specification, the "sequence identity" of a base sequence can be determined using methods and sequence analysis software well known to those skilled in the art. Examples include the blastn program of the BLAST algorithm and the fasta program of the FASTA algorithm. In the present invention, the "sequence identity" of a base sequence to be evaluated with base sequence X is a value expressed as a percentage of the frequency at which the same base appears in the same location in the base sequence, including the gap portion, when base sequence X and the base sequence to be evaluated are aligned and gaps are introduced as necessary to maximize the degree of base agreement between the two.

[0018] Of the outer membrane formation-related genes mentioned above, arcA is the gene for expressing ArcA (Aerobic respiration control). An example of the nucleotide sequence of the structural gene encoding ArcA in E. coli is shown in SEQ ID NO: 4. The signal sequence of arcA is shown in SEQ ID NO: 5.

[0019] bamD is the gene for expressing the outer membrane protein BamD. An example of the nucleotide sequence of the structural gene encoding BamD in E. coli is shown in SEQ ID NO: 6. The signal sequence of bamD is shown in SEQ ID NO: 7.

[0020] cyoA is the gene for expressing CyoA, a membrane protein that makes up the respiratory system. An example of the nucleotide sequence of the structural gene encoding CyoA in E. coli is shown in SEQ ID NO: 8. The signal sequence of cyoA is shown in SEQ ID NO: 9.

[0021] dppA is the gene for expressing DppA, a dipeptide-binding protein. An example of the nucleotide sequence of the structural gene encoding DppA in E. coli is shown in SEQ ID NO: 10. The signal sequence of dppA is shown in SEQ ID NO: 11.

[0022] ecnB is the gene for expressing the toxin lipoprotein EcnB. An example of the nucleotide sequence of the structural gene encoding EcnB in E. coli is shown in SEQ ID NO: 12. The signal sequence of ecnB is shown in SEQ ID NO: 13.

[0023] lpp is the gene for expressing the major outer membrane lipoprotein Lpp. An example of the nucleotide sequence of the structural gene encoding Lpp in E. coli is shown in SEQ ID NO: 14. The signal sequence of lpp is shown in SEQ ID NO: 15.

[0024] mepS is the gene for expressing MepS, a peptidoglycan DD-endopeptidase / peptidoglycan LD-peptidase. An example of the nucleotide sequence of the structural gene encoding MepS in E. coli is shown in SEQ ID NO: 16. The signal sequence of mepS is shown in SEQ ID NO: 17.

[0025] mrcA is the gene for expressing MrcA, a peptidoglycan glycosyltransferase / peptidoglycan DD-transpeptidase. An example of the nucleotide sequence of the structural gene encoding MrcA in E. coli is shown in SEQ ID NO: 18. The signal sequence of mrcA is shown in SEQ ID NO: 19.

[0026] mrcB is the gene for expressing MrcB, a peptidoglycan glycosyltransferase / peptidoglycan DD-transpeptidase. An example of the nucleotide sequence of the structural gene encoding MrcB in E. coli is shown in SEQ ID NO: 20. The signal sequence of mrcB is shown in SEQ ID NO: 21.

[0027] nlpI is the gene for expressing the lipoprotein NlpI. An example of the nucleotide sequence of the structural gene encoding NlpI in E. coli is shown in SEQ ID NO: 22. The signal sequence of NlpI is shown in SEQ ID NO: 23.

[0028] ompA is the gene for expressing the outer membrane protein OmpA. An example of the nucleotide sequence of the structural gene encoding OmpA in E. coli is shown in SEQ ID NO: 24. The signal sequence of OmpA is shown in SEQ ID NO: 25.

[0029] oppA is the gene for expressing the periplasmic oligopeptide-binding protein OppA. An example of the nucleotide sequence of the structural gene encoding OppA in E. coli is shown in SEQ ID NO: 26. The signal sequence of oppA is shown in SEQ ID NO: 27.

[0030] pal is the gene for expressing the outer membrane lipoprotein Pal. An example of the nucleotide sequence of the structural gene encoding Pal in E. coli is shown in SEQ ID NO: 28. The signal sequence of pal is shown in SEQ ID NO: 29.

[0031] slyB is the gene for expressing SlyB, a peptidyl-prolyl cis-trans isomerase. An example of the nucleotide sequence of the structural gene encoding SlyB in E. coli is shown in SEQ ID NO: 30. The signal sequence of slyB is shown in SEQ ID NO: 31.

[0032] tolA is the gene for expressing TolA, one of the proteins that make up the Tol / Pal system. The Tol / Pal system is a complex of multiple proteins found in Gram-negative bacteria, and has been reported to be involved in outer membrane invagination during cell division and maintenance of outer membrane structure. An example of the nucleotide sequence of the structural gene encoding TolA in E. coli is shown in Sequence ID No. 32.

[0033] 1-3 Escherichia coli strains with modified outer membrane formation-related genes In this specification, "modification" of a gene refers to a change in the base sequence of a naturally occurring gene (also called the "wild type"). The term "modification" is not particularly limited as long as some change is made to the base sequence of the original gene, but examples include complete deletion or partial mutation.

[0034] The genetically modified Escherichia coli strain used in the method of the present invention has a genetic modification that alters the expression of one or more outer membrane formation-related genes. Here, "a genetic modification that alters the expression of one or more outer membrane formation-related genes" means a genetic modification in which the activity of the protein encoded by the gene is significantly altered compared to the parent strain. In particular, a genetic modification that weakens the activity of the protein encoded by the gene compared to the parent strain is preferred, and this includes a genetic modification in which the activity is completely eliminated. A state of weakened protein activity refers to a state in which the expression level of mRNA, the transcript of the target gene, or the protein, the translation product, is reduced, or a state in which the mRNA, the transcript of the target gene, or the protein, the translation product, does not function normally as mRNA or protein.

[0035] In this specification, "deletion" of a gene means deletion or damage, preferably deletion. Examples of partial mutations include deletions, insertions, and substitutions, but preferably partial deletions or partial substitutions. "Partial deletion" means partially deleting a gene or protein. "Partial substitution" means that a part of the base sequence of a gene or the amino acid sequence of a protein is replaced with another sequence, but in the present invention, a partial substitution that causes a missense mutation is preferred. Hereinafter, the "one or more outer membrane formation-related genes" to be modified will also simply be referred to as the "target gene." The target gene may be one outer membrane formation-related gene, or it may be two or more outer membrane formation-related genes. Furthermore, multiple locations of one outer membrane formation-related gene may be modified.

[0036] When a partial deletion is made by deleting a portion of the coding region of the amino acid sequence of the protein encoded by the target gene, any region such as the N-terminal region, internal region, or C-terminal region may be deleted. Furthermore, it is preferable that the sequences before and after the deleted region do not have matching reading frames. Preferably, in the genomic DNA, at least a portion of the coding region and / or expression regulatory sequence of the target gene can be deleted, for example, a region consisting of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the total number of bases in the coding region and / or expression regulatory sequence. Alternatively, 100% of the region can be deleted (complete deletion). Also, for example, an E. coli genetically modified strain can be obtained in which at least the region from the start codon to the stop codon of the target gene is deleted in the genomic DNA.

[0037] In this specification, the term "expression regulatory sequence" is not particularly limited as long as it is a nucleotide sequence that can be involved in the expression of the coding region, but it may also be a signal sequence and / or SD sequence (e.g., 5'-AGGAGGA-3'). The E. coli genetically modified strains of the present invention include modified strains having a complete deletion or partial mutation of the signal sequence and / or SD sequence.

[0038] Other examples of modifications to the target gene in the genomic DNA of Escherichia coli strains include introducing missense mutations, introducing stop codons (nonsense mutations), or introducing frameshift mutations that add or delete one or two bases into the amino acid sequence coding region of the gene on the genomic DNA.

[0039] Another example of modifying the target gene in the genomic DNA of an Escherichia coli strain is achieved by inserting another sequence into the gene's expression regulatory sequence or amino acid sequence coding region on the genomic DNA. The insertion site may be in any region of the gene. Furthermore, it is preferable that the sequences before and after the insertion site do not have matching reading frames. The other sequence is not particularly limited, but examples include marker genes. Examples of marker genes include, but are not limited to, drug resistance markers for drugs such as kanamycin, ampicillin, tetracycline, and chloramphenicol, and nutritional requirement markers for nutrients such as leucine, histidine, lysine, methionine, arginine, tryptophan, and uracil.

[0040] The aforementioned genetically modified E. coli strains can be achieved, for example, through mutation, genetic recombination technology, gene expression suppression using RNAi, gene editing, etc.

[0041] Mutagenesis treatments include ultraviolet irradiation or treatment with mutagens commonly used in mutagenesis, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0042] For genetic engineering techniques, known techniques can be used (e.g., FEMS Microbiology Letters 165(1998),335-340, JOURNAL OF BACTERIOLOGY,Dec.1995,p7171-7177, Curr Genet 1986;10(8):573-578, WO98 / 14600, etc.).

[0043] A suitable method can also be used to prepare a partially mutant E. coli outer membrane formation-related gene and integrate it into the genome of any E. coli strain. Methods for preparing a partially mutant E. coli outer membrane formation-related gene include methods known to those skilled in the art, such as overlap PCR using appropriate oligonucleotides as primers or total synthesis. Methods for integrating the prepared partially mutant E. coli outer membrane formation-related gene into host cells can be appropriately described using known methods, such as the Red-recombinase system (Datsenko KA, and Wanner BL (2000), Proc. Natl. Acad. Sci. USA, 97(12), 6640-6645) or methods involving integration by the homologous recombination mechanism of the host cell (Link AJ et al. (1997), J. Bacteriol., 179, 6228-6237). In particular, the method described by Link et al., which does not involve the integration of foreign genes other than the E. coli genome into the genome, is preferred.

[0044] In this context, methods for preparing recombinant vectors of outer membrane formation-related genes with partial mutations include, but are not limited to, ligation, in-fusion (Clontec), PCR, and total synthesis. Furthermore, to introduce the recombinant vector into host cells, for example, when using E. coli as the host, methods such as the calcium chloride method or electroporation can be used, but are not limited to these.

[0045] To increase the production and secretion of foreign proteins, it is also possible to combine the co-expression of periplasmic chaperones such as FkpA, Dsb, and SurA in partial mutant strains.

[0046] Modification of a target gene in the genomic DNA of an E. coli strain can also be achieved by replacing the gene in the E. coli strain's genomic DNA with a deletion gene or a marker gene. Here, a deletion gene is an inactive gene that has been modified to not produce a normally functioning protein by deleting part or all of the target gene. Examples of deletion genes include, for example, linear DNA containing an arbitrary sequence that does not have the function of the target gene, and having the upstream and downstream sequences of the site to be replaced on the genomic DNA (i.e., part or all of the target gene) at both ends of the arbitrary sequence, or linear DNA in which the upstream and downstream sequences of the site to be replaced on the genomic DNA are directly connected. Examples of marker genes include, for example, linear DNA containing a marker gene sequence, and having the upstream and downstream sequences of the site to be replaced on the genomic DNA (i.e., part or all of the target gene) at both ends of the marker gene sequence. By transforming the E. coli strain with the linear DNA described above, homologous recombination can be induced upstream and downstream of the target site in the host strain's genomic DNA, thereby replacing the target site with the linear DNA sequence in a single step. This substitution can cause the target gene to be deleted in the genomic DNA of the E. coli strain.

[0047] The marker gene may be removed after the above substitution if necessary. For this purpose, it is preferable to add homologous recombination sequences or flippase recognition target (FRT) sequences to both ends of the marker gene so that it can be removed efficiently.

[0048] When a genetically modified E. coli strain is a strain in which the pal gene has been modified, it includes E. coli strains in which the gene encoding Pal (having the amino acid sequence shown in SEQ ID NO: 33) on the E. coli genome (SEQ ID NO: 40) has been mutated, and E. coli strains in which the gene encoding the signal peptide of Pal (SEQ ID NO: 42) has been mutated. When mutating the signal peptide, other signal peptides such as OmpA, OmpF, and Lpp may also be used.

[0049] The mutation in Pal is preferably a partial mutation, particularly a partial deletion. Escherichia coli containing a partially deleted gene encoding Pal includes Escherichia coli with a partial deletion of the gene encoding Pal (having the amino acid sequence shown in SEQ ID NO: 33) (SEQ ID NO: 28) in the Escherichia coli genome, and Escherichia coli with a partial deletion of the gene encoding the signal peptide of Pal (SEQ ID NO: 34) (SEQ ID NO: 29). The location for partial deletion of Pal can be, for example, the location described in Cascales E. and Lloubes R., (2004), Mol Microbiol., 51(3), 873-885.

[0050] A Pal partial mutant strain can also be prepared by introducing a gene containing a partially mutant Pal-coding gene into a Pal-deficient strain lacking the gene encoding Pal, thereby expressing the partially mutant Pal. However, in the present invention, it is preferable to use Escherichia coli in which Pal (SEQ ID NO: 33) is partially mutant, and it is more preferable that the partial mutant is a partial deletion.

[0051] The partial mutation in Pal is preferably a partial mutation in one or more amino acid sequences selected from the amino acid sequences of positions 3 to 17, 19 to 121, and 123 to 152 of the signal peptide of Pal, SEQ ID NO: 49.

[0052] Examples of E. coli strains with mutations in Pal include: an E. coli strain whose genome contains a gene (sequence number 36) encoding Pal (sequence number 35) with amino acids missing from position 3 to 17; an E. coli strain whose genome contains a gene (sequence number 38) encoding Pal (sequence number 37) with amino acids missing from position 19 to 43; an E. coli strain whose genome contains a gene (sequence number 40) encoding Pal (sequence number 39) with amino acids missing from position 44 to 62; and an E. coli strain whose genome contains a gene (sequence number 42) encoding Pal (sequence number 41) with amino acids missing from position 62 to 93. Examples include E. coli strains, E. coli strains that have a gene (sequence number 44) on their genome that codes for Pal (sequence number 43) with amino acids missing from position 94 to 121, E. coli strains that have a gene (sequence number 46) on their genome that codes for Pal (sequence number 45) with amino acids missing from position 123 to 152, E. coli strains that have a gene (sequence number 48) on their genome that codes for Pal (sequence number 47) with amino acids missing from position 126 to 129, and E. coli strains that have a gene (sequence number 50) on their genome that codes for Pal (sequence number 49) with amino acids missing from position 144 to 147.

[0053] As the Escherichia coli strain used in the method of the present invention, it is preferable to use an Escherichia coli strain containing a pal gene having any of the nucleotide sequences shown in SEQ ID NOs. 36, 38, 40, 42, 44, 46, 48, and 50.

[0054] The Pal having the aforementioned partial mutation may be an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in any of the amino acid sequences shown in SEQ ID NOs. 35, 37, 39, 41, 43, 45, 47, and 49. "One or more" means, for example, 1 to 80, preferably 1 to 70, preferably 1 to 60, preferably 1 to 50, preferably 1 to 40, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, preferably 1 to 5, preferably 1 to 4, preferably 1 to 3, preferably 1 to 2, and preferably 1.

[0055] Pal having the aforementioned partial mutation may have 60% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, and 50. The sequence identity may be 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0056] In this specification, the sequence identity of amino acid sequences can be determined using methods and sequence analysis software well known to those skilled in the art. Examples include the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm. In the present invention, the "sequence identity" of a target amino acid sequence with amino acid sequence X is expressed as a percentage value representing the frequency at which the same amino acid appears in the same location in the amino acid sequence, including the gap portion, when amino acid sequence X and the target amino acid sequence are aligned and gaps are introduced as necessary to maximize the degree of amino acid agreement between the two.

[0057] When a genetically modified E. coli strain is a strain in which the lpp gene has been modified, it includes E. coli in which the gene encoding Lpp (for example, Lpp having the amino acid sequence of SEQ ID NO: 51) on the E. coli genome (for example, the nucleotide sequence of SEQ ID NO: 14) has been mutated, and E. coli in which the gene encoding the signal peptide of Lpp (for example, the nucleotide sequence of SEQ ID NO: 15) has been mutated. When mutating the signal peptide, other signal peptides such as OmpA, OmpF, and Pal may also be used.

[0058] When a genetically modified E. coli strain is a strain in which the ompA gene has been modified, it includes E. coli strains in which the gene encoding OmpA (for example, OmpA having the amino acid sequence of SEQ ID NO: 52) on the E. coli genome has been mutated (for example, the nucleotide sequence of SEQ ID NO: 24), and E. coli strains in which the gene encoding the OmpA signal peptide (for example, the nucleotide sequence of SEQ ID NO: 36) has been mutated. When mutating the signal peptide, other signal peptides such as Lpp, OmpF, and Pal may also be used.

[0059] If a genetically modified E. coli strain is one in which the tolA gene has been modified, it includes E. coli strains in which the gene encoding TolA (for example, TolA having the amino acid sequence shown in SEQ ID NO: 53) on the E. coli genome (for example, the base sequence of SEQ ID NO: 32) has been mutated.

[0060] 1-4 Gene transfer process (process (a)) The method of the present invention includes (a) introducing a gene encoding a target foreign protein into Escherichia coli in which genes related to the maintenance of the outer membrane structure have been modified (hereinafter also referred to as "step (a)").

[0061] 1-4-1 Expression vector The method of the present invention involves introducing a gene encoding a target foreign protein (foreign protein) into a genetically modified strain of E. coli to produce the target foreign protein. More specifically, in the method of the present invention, an expression vector containing a gene encoding the target foreign protein is introduced into the genetically modified strain of E. coli.

[0062] In this specification, "expression vector" means an artificially constructed nucleic acid molecule that has the function of expressing a gene in an expression cassette incorporated into the expression vector in transformed host cells. In addition to the expression cassette, the expression vector may have a cloning site containing one or more restriction enzyme recognition sequences, an overlap region for using Clontec's In-Fusion cloning system, a marker gene such as a drug resistance gene, a self-replicating sequence, etc. Examples of expression vectors include plasmid vectors and artificial chromosomes, but plasmid vectors are preferably used because vector preparation and transformation of E. coli strains are easy. Examples of known plasmids include pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc., but are not limited to these.

[0063] An "expression cassette" consists of a promoter and a gene encoding an exogenous protein, and may also include a terminator. Examples of promoters to be used include, but are not limited to, promoters known to those skilled in the art, such as the lac promoter, tac promoter, ara promoter, tet promoter, and T7 promoter.

[0064] The method of the present invention requires that the target foreign protein be translocated from the cytoplasm of E. coli to the periplasmic space. Therefore, a periplasmic translocation signal sequence must be added to the 5' end of the gene encoding the foreign protein. Examples of periplasmic translocation signals that function in E. coli include, but are not limited to, PelB, OmpA, PhoA, OmpF, and STII. Furthermore, for foreign proteins that require a periplasmic translocation signal for translocation to the periplasmic space, the addition of a periplasmic translocation signal is not required.

[0065] In the method of the present invention, the base sequence of the gene encoding the foreign protein (foreign gene) incorporated into the expression vector is preferably 200 to 4,500 bp in length, and particularly 500 to 2,000 bp in length, in order to ensure the stability of the expression vector and high efficiency of introduction into the host.

[0066] Examples of foreign proteins produced by the method of the present invention include enzymes derived from microorganisms, proteins derived from multicellular organisms such as animals and plants, etc. For example, phytase, protein A, protein G, protein L, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, nuclease, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, and fluorescent proteins, but are not limited to these. Biopharmaceutical proteins are particularly preferred.

[0067] Examples of proteins used in biopharmaceuticals include partial antibodies such as VHH, scFv, and Fab, cytokines, growth factors, protein kinases, protein hormones, Fc fusion proteins, and human serum albumin (HSA) fusion proteins.

[0068] The amino acids that make up the above foreign protein may be natural, unnatural, or modified. Furthermore, the amino acid sequence of the protein may be artificially modified or de-novo designed.

[0069] The drug resistance marker genes of the plasmid vector in the present invention are not particularly limited to the Escherichia coli strains provided in the present invention. Specific examples include kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, and chloramphenicol resistance genes, which can be selected based on resistance in a culture medium containing kanamycin, ampicillin, tetracycline, and chloramphenicol, respectively.

[0070] 1-4-2 Gene transfer In the method of the present invention, the expression vector is introduced into a genetically modified Escherichia coli strain. The method for introducing the expression vector into the Escherichia coli strain is not particularly limited, but can be carried out by gene transfer methods such as electroporation, calcium chloride method, competent cell method, or protoplast method.

[0071] 1-5 Steps for culturing E. coli (Step (b)) The method of the present invention includes a step of culturing a genetically modified strain of Escherichia coli into which the gene encoding the foreign protein obtained in step (a) has been introduced (hereinafter also referred to as "step (b)").

[0072] The genetically modified E. coli strain can be cultured in a suitable culture medium. The culture method may be batch culture, fed-batch culture, or continuous culture. The culture medium may be either a synthetic medium or a natural medium, as long as it contains nutrients necessary for the growth of the genetically modified E. coli strain, such as a carbon source, nitrogen source, inorganic salts, and vitamins.

[0073] Any carbon source that can be utilized by the genetically modified E. coli strain can be used as a carbon source, and examples include carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0074] Examples of nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone.

[0075] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0076] Examples of vitamins include biotin and thiamine. Furthermore, if necessary, substances required for the growth of the genetically modified E. coli strain (for example, required amino acids in the case of an amino acid-requiring strain) can be added.

[0077] In the method of the present invention, a culture medium containing peptone, yeast extract, and sodium chloride is preferably used. The peptone is preferably soy peptone. A preferred example of a culture medium containing peptone, yeast extract, and sodium chloride is 2×YT medium.

[0078] In the method of the present invention, the culture conditions are not particularly limited, but preferably examples include shaking culture and stirring culture. The culture temperature is 20 to 50°C, preferably 20 to 42°C, and more preferably 25 to 33°C. The culture time is 3 hours to 5 days, preferably 5 hours to 3 days.

[0079] In the method of the present invention, nutrients or inducers can be supplied to the culture medium by shot or flow-add, as needed.

[0080] The inducing substance is appropriately selected depending on the promoter used. For example, lactose or isopropyl-β-thiogalactopyranoside (IPTG) are used for direct induction of the lac promoter or tac promoter, or for indirect induction of the T7 promoter by expressing T7 RNA polymerase with the lac promoter. When using IPTG as an inducing substance, protein expression can be induced by adding it to a final concentration of 0.1 to 2.0 mM, more preferably 0.2 to 1.0 mM.

[0081] Furthermore, the timing of inducing foreign protein expression is not particularly limited as long as the foreign protein is expressed and secreted into the culture supernatant, but it is preferably in the early, mid, or late stages of the logarithmic growth phase, and particularly preferably in the late stages of the logarithmic growth phase.

[0082] 1-6 Step to recover the target protein (Step (c)) The method of the present invention includes a step of recovering the target foreign protein from the periplasmic fraction of the genetically modified Escherichia coli strain cultured in step (b) (hereinafter also referred to as "step (c)").

[0083] 1-6-1 Recovery of bacterial cells Any known method, such as centrifugation, can be used to recover the bacterial cells from the culture medium. In this case, to further improve the yield of the target protein, the culture supernatant may be recovered and the target protein may be purified separately from the bacterial cells.

[0084] 1-6-2 Extraction of the periplasmic fraction The target protein is recovered from the recovered bacterial cells. To suppress contamination by cytoplasmic proteins, the target protein must be extracted from the periplasmic fraction of the bacterial cells without cell disruption. Any known method can be used to extract the protein from the periplasmic fraction in step (c).

[0085] Known methods for extracting proteins from the periplasmic fraction of E. coli include, for example, osmotic shock, chelating agent treatment, treatment with cell wall-degrading enzymes such as lysozyme, treatment with weak chaotropic substances (e.g., arginine) (see, for example, Patent Document 1), heat treatment, low-concentration surfactant treatment, and freeze-thaw cycles. These methods are preferable in terms of the ratio of target protein to impurities in the extract, provided that the conditions for extracting proteins from the periplasmic fraction without exposing the cytoplasmic fraction to the extracellular space due to excessive cell disruption are met, but they are not particularly limited and any of them can be used. These methods may be used individually or in combination. To reduce the risk of cytoplasmic exposure due to E. coli cell disruption, it is preferable to use chemical methods, particularly cold osmotic shock using osmotic shock, methods using chelating agents, and methods using low-concentration surfactants, rather than mechanical methods such as sonication or high-pressure homogenizers. Generally, the yield of target proteins by such chemical methods is known to be lower than that by mechanical methods. The method of the present invention, by using genetically modified E. coli strains, makes it possible to obtain the target protein in high yield even by chemical methods.

[0086] The cold osmotic shock method is the most common technique used for extracting the periplasmic fraction, and for example, the following procedure can be used: Suspend the bacterial cells in a sucrose hypertonic solution prepared with EDTA / Tris buffer, collect the cells, and suspend them in ice-cold water. Collect the cells again, and the supernatant is obtained as the periplasmic fraction. Here, the bacterial cells may be washed with Tris buffer before suspending them in the hypertonic solution. Also, the suspension of bacterial cells in the hypertonic solution and collection may be performed in multiple cycles.

[0087] As an example of a method using a chelating agent, the following method can be employed: Bacterial cells are suspended in a weakly basic Tris / EDTA buffer containing a relatively high concentration of EDTA and incubated at room temperature for 15 minutes to 6 hours. The cells are collected again, and the supernatant is obtained as the periplasm fraction. The suspension of bacterial cells in Tris / EDTA buffer and collection may be repeated. In this specification, the above method is also referred to as the "Tris / EDTA method".

[0088] As a method using low concentrations of surfactants, for example, a method using bile salts such as sodium cholate and sodium deoxycholate, which are less likely to cause protein denaturation, can be employed. Specifically, for example, the following method can be used: Bacterial cells are suspended in a 0.15% by weight sodium deoxycholate solution, shaken at room temperature for 30 minutes to 7 hours, then collected, and the supernatant is obtained as the periplasm fraction. The suspension of bacterial cells in the sodium deoxycholate solution and collection can be performed multiple times.

[0089] When using the known periplasm extraction methods described above, if a typical E. coli strain is used, the periplasm fraction may not contain a sufficient amount of protein depending on the type of target protein. The method of the present invention has the advantage of being able to include a larger amount of the target protein in the periplasm fraction.

[0090] 1-6-3 Protein Purification The target protein can be purified from the periplasm fraction. Any known method can be used for protein purification. For example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, etc., can be used. The method of the present invention does not involve cell disruption, making it possible to efficiently purify the target protein from a state with minimal contamination from other proteins.

[0091] The protein may be purified from the culture supernatant separately from, or mixed with, the periplasm fraction. Recovering the protein from the culture supernatant can further increase the protein yield.

[0092] 2. Method for preparing host E. coli The method for producing host Escherichia coli for the production of the protein of the present invention (hereinafter also referred to as "the method for producing host Escherichia coli of the present invention") is characterized by comprising the following steps (i) and (ii). (i) A step of modifying genes related to the maintenance of the outer membrane structure of Escherichia coli; and (ii) The process of introducing a gene encoding the target foreign protein into the E. coli whose genes have been modified in (i).

[0093] The Escherichia coli produced by the host Escherichia coli production method of the present invention expresses the target foreign protein through the introduced gene. In this case, it is preferable that the target foreign protein accumulates in the periplasm of the Escherichia coli.

[0094] 2-1 Genetic modification process of host E. coli (Step (i)) The present invention provides a method for producing host Escherichia coli, which includes a step of modifying the outer membrane formation-related genes of Escherichia coli (hereinafter also referred to as "step (i)"). The Escherichia coli strain used in the present invention's method for producing host Escherichia coli is as described in "1-1 Escherichia coli strain" of "1 Protein Production Method" above. The outer membrane formation-related genes to be modified are as described in "1-2 Outer membrane formation-related genes" above. Furthermore, the specific methods and conditions for modifying the outer membrane formation-related genes of Escherichia coli are as described in "1-3 Escherichia coli strain with modified outer membrane formation-related genes" above.

[0095] 2-2 Foreign gene introduction process (process (ii)) The present invention provides a method for producing a host Escherichia coli, which includes a step (hereinafter also referred to as "step (ii)") of introducing a gene encoding a target foreign protein into the genetically modified Escherichia coli strain obtained in step (i). In step (ii), the expression vector used for gene introduction, the introduction method, etc., are as described in "1-4 Gene Introduction Step (Step (a))" above. [Examples]

[0096] The present invention will be described in more detail with reference to the following embodiments, but the present invention is not limited to these embodiments.

[0097] The amino acid numbers described in this invention refer to the numbers in the entire protein, including the signal sequence. The genetic engineering techniques used in this invention, such as polymerase chain reaction (PCR), gene synthesis, DNA isolation and purification, restriction enzyme treatment, cloning of modified DNA, and transformation, are known to those skilled in the art. Unless otherwise specified, the following examples were carried out according to the procedures described in the accompanying manuals of the reagent and equipment manufacturers.

[0098] In the following examples, PCR was performed using Prime STAR HS DNA Polymerase (Takara Bio Inc.). Purification of PCR products and restriction enzyme reactions was performed using the QIAuick Gel Extraction Kit (QIAGEN). DNA fragments were prepared using conventional gene synthesis methods other than PCR. Plasmid vectors used for transformation were prepared by introducing the constructed vector into E. coli DH5α competent cells (Takara Bio Inc.), culturing the resulting transformants, and amplifying the resulting cells. Plasmid extraction from plasmid-carrying strains was performed using the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.). The cloning vector used was a modified version of pTH18cs (National Institute of Genetics: NIG), containing a tetracycline resistance gene as a marker. An expression vector containing the lac repressor gene was used for the expression of the target protein.

[0099] (Example 1) Preparation of a genetically modified Escherichia coli strain related to outer membrane formation Total pal deletion, partial pal deletion, and lpp point mutant strains of *Escherichia coli* were prepared using the method of Link AJ et al. (J. Bacteriol., 1997, 179, 6228-6237). To completely delete the pal gene, a DNA fragment (SEQ ID NO: 1) with EcoRI and SalI cleavage sites at both ends and homology arms of approximately 500 bp each upstream and downstream of the pal gene was amplified by PCR. The DNA fragment and cloning vector prepared above were cleaved with restriction enzymes EcoRI and SalI, and ligation was performed using each cleaved DNA fragment. The plasmid (pΔpal) thus prepared was introduced into *Escherichia coli* BL21 strain using the calcium chloride method, and pΔpal-containing strains were selected using a medium containing tetracycline. Following the method of Link AJ et al., deletion of the target gene by homologous recombination was confirmed, and the resulting modified strain was designated as the Δpal strain.

[0100] Using the same procedure as described above, DNA fragments (SEQ ID NO: 2) with approximately 200 bp homology arms upstream and downstream of the target modified pal gene, which has EcoRI and SalI cleavage sites at both ends and a deletion from positions 39 to 63 of Pal, and DNA fragments (SEQ ID NO: 3) with approximately 200 bp homology arms upstream and downstream of the target modified lpp gene, which has Gly at position 14 of the Lpp signal sequence replaced with Asp, were amplified, and cloning vectors (denoted as "ppal1" and "plpp1," respectively) were prepared. Similar to pΔpal, ppal1 and plpp1 were introduced into E. coli strain BL21, and modified strains were selected using a medium containing tetracycline. After confirming that the target gene had been modified by homologous recombination, the obtained modified strains were named pal1 and lpp1, respectively.

[0101] (Example 2) Measurement of the efficiency of target protein recovery from gene-modified Escherichia coli strains related to outer membrane formation. This example was conducted to investigate the recovery efficiency of the target protein from the wild-type strain and three genetically modified E. coli strains related to outer membrane formation prepared in Example 1. In this example, various E. coli strains were made to produce anti-von Willebrand factor (vWF) VHH antibody as the target protein, and its recovery efficiency was investigated. E. coli strains into which an expression vector containing the gene encoding the anti-vWF VHH antibody (SEQ ID NO: 54) was introduced by electroporation were cultured in 1 mL of semi-synthetic medium. The total amount of anti-vWF nanobodies (target protein) contained in the soluble fraction of the bacterial cells was taken as 100% recovery, and the recovery efficiency of the target protein recovered from the periplasm fraction of each E. coli strain was calculated. Each fraction was prepared using the following two methods.

[0102] (1) Cold osmotic shock method 250 μL of culture medium was centrifuged at 6,400 × g for 10 minutes to separate the supernatant from the cells. The cells were suspended in 250 μL of hypertonic solution (100 mM Tris / HCl, 500 mM sucrose, 0.5 mM EDTA, pH 8.0), allowed to stand at room temperature for 10 minutes, then centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as hypertonic fraction 1. The cells were washed by resuspending them in 250 μL of hypertonic solution, and centrifuged at 12,000 × g for 5 minutes. The supernatant was collected as hypertonic fraction 2, and the cells were suspended in 250 μL of ice-cold hypotonic solution (1 mM MgCl2). The suspension was centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as hypotonic fraction 1. Cells were suspended in 250 μL of hypotonic solution and centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as hypotonic fraction 2. Cells were suspended in 250 μL of PBS and lysed twice for 30 seconds each using an ultrasonic disruptor (UH-50: SMT Co.), then centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as lysation fraction 1. Cells were suspended in 250 μL of PBS and washed, then centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as lysation fraction 2.

[0103] (2) Tris / EDTA method (1) Similarly, 250 μL of culture medium was centrifuged at 6,400 × g for 10 minutes to separate the supernatant from the cells. The cells were suspended in 250 μL of Tris / EDTA buffer (100 mM Tris / HCl, 10 mM EDTA pH 7.4), allowed to stand at room temperature for 90 minutes, then centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as T / E fraction 1. The cells were washed by resuspending them in 250 μL of Tris / EDTA buffer and centrifuged at 12,000 × g for 5 minutes. The supernatant was collected as T / E fraction 2. The cells were suspended in 250 μL of PBS, lysed twice for 30 seconds each using an ultrasonic disruptor (UH-50: SMT), centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as lysation fraction 1. The cells were suspended in 250 μL of PBS and washed, then centrifuged at 12,000 × g for 5 minutes, and the supernatant was collected as fraction 2.

[0104] Western blotting analysis: 6 μL of each fraction recovered by the cold osmotic shock method and Tris / EDTA method described above was mixed with 2 μL of 4×Laemmli Sample Buffer (Biorad), heated at 95°C for 5 minutes, and then 3 μL was applied to a polyacrylamide gel. After SDS-PAGE, the mixture was blotted onto a PVDF membrane and blocked with Blocking One (Nacalai Tesque). After washing with TBS-T, antibody reactions were performed using anti-humanized VHH polyclonal rabbit antibody and anti-rabbit polyclonal goat antibody-HRP, detected using EzWestLumi plus (ATTO), and band intensity was determined using ChemiDoc (Biorad) and Quantity One (Biorad). The recovery rate of each fraction was calculated from the band intensity. In the cold osmotic shock method, the combined amount of hypertonic fractions 1 and 2, and hypotonic fractions 1 and 2 was used as the recovery amount. In the Tris / EDTA method, the combined amount of T / E fractions 1 and 2 was used as the recovery amount. The ratio of the aforementioned recovery amount to the total anti-vWF nanobodies (target protein) from the bacterial cells, including those generated from lysated fractions 1 and 2 (recovery efficiency), was calculated, and the differences between wild-type strains and genetically modified E. coli strains related to outer membrane formation were investigated (Table 1). When comparing wild-type strains and genetically modified E. coli strains related to outer membrane formation, the recovery rate improved in both the cold osmotic shock method and the Tris / EDTA method when using strains with modified pal and lpp.

[0105] [Table 1]

[0106] In all strains, secretion of a portion of the target protein was observed in the culture supernatant (data not shown). It was found that the amount of target protein recovered could be further improved by further recovering the target protein from the culture supernatant.

[0107] (Example 3) Measurement of the efficiency of target protein recovery from gene-modified E. coli strains related to outer membrane formation using the Keio collection. This example was conducted to investigate the recovery efficiency of target proteins from genetically modified E. coli strains related to outer membrane formation, using the Keio collection, a single-gene deletion library of E. coli. In this example, anti-vWF nanobodies were produced as the target protein in the Keio collection, which consists of mutant strains of E. coli BW25113, specifically those lacking ompA, tolA, mepS, nlpI, arcA, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB, and their recovery efficiency was investigated. An expression vector containing the gene encoding the anti-vWF nanobodies (SEQ ID NO: 54) was introduced into each mutant strain by electroporation, and the strains were cultured in 0.8 mL of semi-synthetic medium. For each mutant strain, the total amount of anti-vWF nanobodies (target protein) contained in the soluble fraction of the bacterial cells was set as 100% recovery, and the recovery efficiency of the target protein recovered from the periplasm fraction was calculated. Each fraction was prepared by cold osmotic shock under the same conditions as in Example 2. Furthermore, the anti-vWF nanobodies in each fraction were analyzed by Western blotting under the same conditions as in Example 2.

[0108] The recovery rate of anti-vWF nanobodies in each fraction was calculated from the band intensity. The total recovery amount was calculated by combining the hypertonic fractions 1 and 2 and the hypotonic fractions 1 and 2. The proportion of the recovered amount to the total anti-vWF nanobodies from the bacterial cells, including those generated from the lysated fractions 1 and 2, was calculated, and the difference between the wild-type strain (BW25113 strain) and the genetically modified E. coli strains related to outer membrane formation was examined (Table 2). When comparing the wild-type strain and the genetically modified E. coli strains related to outer membrane formation, it was confirmed that using knockout strains of ompA, tolA, mepS, nlpI, arcA, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB improved the recovery rate by cold osmotic shock in all genetically modified E. coli strains related to outer membrane formation.

[0109] [Table 2] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A method for producing protein, comprising the following steps (a) to (c): (a) A step of introducing a gene encoding a target foreign protein into Escherichia coli in which genes related to the maintenance of the outer membrane structure have been modified, wherein the gene related to the maintenance of the outer membrane structure is at least one selected from the group consisting of pal, lpp, ompA, tolA, mepS, nlpI, bamD, cyoA, ecnB and slyB; (b) The step of culturing the E. coli in (a); and (c) A step to recover the target foreign protein from the periplasmic fraction of the cultured bacterial cells.

2. The method according to claim 1, wherein the aforementioned modification is completely absent.

3. The method according to claim 1 or 2, wherein the modification is a partial mutation.

4. The method according to claim 3, wherein the modification is a partial mutation of the signal sequence.

5. The method according to claim 3, wherein the modification is a partial mutation of a structural gene.

6. The method according to any one of claims 1 to 5, wherein the Escherichia coli is derived from strain B or strain K12.

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