Recombinant cells, expression constructs, and methods for producing recombinant proteins

Abstract

To provide a recombinant cell with an increased expression level of recombinant protein.SOLUTION: A recombinant cell has: a first expression cassette including a polynucleotide sequence coding recombinant protein which is connected operably to a first promoter; and a second expression cassette including a polynucleotide sequence coding the recombinant protein which is connected operably to a second promoter different from the first promoter.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to recombinant cells, expression constructs and methods for producing recombinant proteins. [Background technology]

[0002] Many proteins needed for research, industrial, or medical applications (e.g., enzymes, vaccines, structural proteins, hormones, and biopharmaceutical proteins) are produced industrially using recombinant cells. For example, bacterial cells (especially E. coli cells) grow rapidly to high cell densities in inexpensive media, making them particularly suitable for the large-scale production of recombinant proteins.

[0003] To produce large amounts of recombinant proteins, several aspects must be considered when designing an expression cassette containing a polynucleotide sequence encoding the protein and a sequence (e.g., a promoter) that controls its expression. For example, in host cells used for recombinant protein production, codon optimization of the polynucleotide sequence encoding the target protein has been attempted. Other approaches include placing the transcription of the polynucleotide sequence encoding the target protein under the control of a strong promoter and an effective terminator, or optimizing translation by introducing an appropriate ribosome binding site. Furthermore, attempts have been made to increase the copy number of the polynucleotide sequence in the host cell.

[0004] Furthermore, when an expression cassette is incorporated into a plasmid, there is a problem that it is lost during cell division of the host cell, so efforts have been made to incorporate the expression cassette into the genomic DNA of the host cell by homologous recombination or the like (e.g., Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5996052 Summary of the Invention [Problem to be solved by the invention]

[0006] However, high expression of recombinant proteins by foreign genes is detrimental to the survival of host cells. For example, when many copies of a foreign gene are produced in a host cell, this leads to a shortage of resources for protein expression (RNA and protein raw materials, intracellular machinery for transcription and translation), and increases the metabolic burden on the host cell. As a result, the yield of the recombinant protein is suppressed. That is, after the yield of the recombinant protein reaches a certain amount, the productivity of the recombinant protein gradually decreases. Therefore, maintaining sustainable productivity in recombinant cells is a major challenge in recombinant protein production (especially in long-term recombinant protein production using continuous culture or fed-batch culture methods).

[0007] An object of the present invention is to provide a recombinant cell that expresses an increased amount of a recombinant protein. Another object of the present invention is to provide an expression construct that expresses an increased amount of a recombinant protein. [Means for solving the problem]

[0008] The present invention relates to, for example, the following inventions. [1] A recombinant cell having a first expression cassette comprising a polynucleotide sequence encoding a recombinant protein operably linked to a first promoter, and a second expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a second promoter different from the first promoter. [2] The recombinant cell according to [1], wherein the transcriptional activity of the second promoter is weaker than the transcriptional activity of the first promoter. [3] The recombinant cell according to [1] or [2], wherein the polynucleotide sequences of the first promoter and the second promoter have at least 80% sequence identity. [4] The recombinant cell according to any one of [1] to [3], wherein the first promoter is a T7 promoter. [5] The recombinant cell according to any one of [1] to [4], wherein the second promoter comprises a base sequence selected from the base sequence shown in SEQ ID NO: 2 and the base sequence shown in SEQ ID NO: 3. [6] The recombinant cell according to any one of [1] to [5], further comprising a third expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a third promoter. [7] The recombinant cell according to [6], wherein the third promoter is the same as the first promoter or the second promoter. [8] The recombinant cell according to any one of [1] to [7], wherein the recombinant protein is fibroin. [9] The recombinant cell according to any one of [1] to [8], wherein the first expression cassette and the second expression cassette are integrated into host genomic DNA.

[10] An expression construct having a first expression cassette comprising a polynucleotide sequence encoding a recombinant protein operably linked to a first promoter, and a second expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a second promoter different from the first promoter.

[11] The expression construct according to

[10] , wherein the transcriptional activity of the second promoter is weaker than the transcriptional activity of the first promoter.

[12] The expression construct according to

[10] or

[11] , wherein the polynucleotide sequences of the first promoter and the second promoter have at least 80% sequence identity.

[13] The recombinant cell according to any one of

[10] to

[12] , wherein the first promoter is a T7 promoter.

[14] The recombinant cell according to any one of

[10] to

[13] , wherein the second promoter comprises a base sequence selected from the base sequence shown in SEQ ID NO: 2 and the base sequence shown in SEQ ID NO: 3.

[15] The expression construct according to any one of

[10] to

[14] , further comprising a third expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a third promoter.

[16] The expression construct according to

[15] , wherein the third promoter is the same as the first promoter or the second promoter.

[17] A method for producing the above-mentioned recombinant protein, comprising a production step of culturing in a protein production medium a recombinant cell having a first expression cassette containing a polynucleotide sequence encoding the recombinant protein operably linked to a first promoter, and a second expression cassette containing a polynucleotide sequence encoding the recombinant protein operably linked to a second promoter different from the first promoter.

[18] The method according to

[17] , wherein the recombinant cells are cultured by continuous culture or fed-batch culture in the production process.

[19] The method according to

[17] or

[18] , wherein the production process includes an expression induction step of inducing expression of the recombinant protein by adding an expression inducer to the protein production medium.

[20] The production method according to any one of

[17] to

[19] , wherein the production step includes adding an expression inducer to the protein production medium to induce expression of the recombinant protein, followed by culturing for 9 hours or more. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a recombinant cell that expresses an increased amount of a recombinant protein. Also, according to the present invention, it is possible to provide an expression construct that expresses an increased amount of a recombinant protein. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a graph showing the results of measuring the transcriptional activity of the T7 promoter, T7.5 promoter, T7.51 promoter, and SPT3 promoter by quantitative PCR. [Figure 2] FIG. 1 is a schematic diagram showing an outline of a method for integrating a modified fibroin expression cassette into a host chromosome by utilizing the lysogenization mechanism of HK022 phage. [Figure 3] FIG. 1 is a schematic diagram showing an outline of a method for integrating a modified fibroin expression cassette into a host chromosome by utilizing the lysogenization mechanism of φ80 phage. [Figure 4] FIG. 1 is a schematic diagram showing an outline of a method for integrating a modified fibroin expression cassette into a host chromosome using the homologous recombination system of λ phage. [Figure 5] 1 is a graph showing the results of evaluating the protein (modified fibroin) production amounts of the T7-T7-T7 strain and the T7-T7.51-T7.51 strain. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0012] [Recombinant cells] The recombinant cell of this embodiment has a first expression cassette comprising a polynucleotide sequence encoding a recombinant protein operably linked to a first promoter, and a second expression cassette comprising a polynucleotide sequence encoding a recombinant protein operably linked to a second promoter different from the first promoter.

[0013] (First Expression Cassette) The first expression cassette comprises at least a first promoter and a polynucleotide sequence encoding a recombinant protein operably linked to the first promoter. The first expression cassette may further comprise one or more regulatory sequences operably linked to the first promoter and the polynucleotide sequence encoding the recombinant protein.

[0014] Regulatory sequences are sequences that control the expression of a recombinant protein (target protein) in a host (e.g., enhancers, ribosome binding sequences, transcription termination sequences, etc., in addition to promoters), and can be selected appropriately depending on the type of host. Regulatory sequences may be exogenous or endogenous (host-derived regulatory sequences).

[0015] The first promoter can be a promoter that functions in the host used, depending on the host. Specific examples of promoters are described below. The promoter used in the present invention may be a naturally occurring one or an artificially produced one. In the present invention, promoters having at least two different transcriptional activities are used, and these promoters may be of different origins or may be of the same origin.

[0016] (Second expression cassette) The second expression cassette comprises at least a second promoter and a polynucleotide sequence encoding a recombinant protein operably linked to the second promoter. The second promoter is different (has a different nucleotide sequence) from the first promoter. The recombinant protein expressed by the second expression cassette is preferably the same as the recombinant protein expressed by the first expression cassette.

[0017] The second expression cassette may further comprise one or more regulatory sequences operably linked to the second promoter and the polynucleotide sequence encoding the recombinant protein, the regulatory sequences being as described above.

[0018] The second promoter can be a promoter that functions in the host depending on the host used. Specific examples of promoters are described below.

[0019] (First and second promoters) The second promoter preferably has a transcription activity weaker than that of the first promoter. The transcription activity of the second promoter is preferably 50 or less, more preferably 25 or less, even more preferably 10 or less, even more preferably 5 or less, and particularly preferably 1 or less, when the transcription activity of the first promoter is taken as 100. The transcription activity of the promoter can be measured by the method described in the Examples below.

[0020] In one embodiment of the present invention, the first promoter and the second promoter may be in the relationship of a commonly used promoter and its mutant promoter, or in the relationship of a wild-type promoter and its mutant promoter.

[0021] A mutant promoter refers to a promoter having a polynucleotide sequence in which one or more residues have been substituted, deleted, inserted, and / or added compared to the polynucleotide sequence of a commonly used promoter or a wild-type promoter. Mutant promoters can be prepared, for example, by genetic engineering techniques.

[0022] In one embodiment of the invention, the polynucleotide sequence of the first promoter and the polynucleotide sequence of the second promoter may have at least 80% sequence identity, may have 85% or more sequence identity, may have 90% or more sequence identity, may have 95% or more sequence identity, may have 98% or more sequence identity, or may have 99% or more sequence identity.

[0023] In one embodiment of the present invention, the first promoter may be a promoter that has strong transcriptional activity in a host cell, and the second promoter may be a mutant promoter of the first promoter with attenuated transcriptional activity. Promoters that have strong transcriptional activity in a host cell are known to those skilled in the art, and examples thereof include the T7 promoter (SEQ ID NO: 1) when a prokaryotic cell such as Escherichia coli is used as the host. Examples of the second promoter include the T7.5 promoter (SEQ ID NO: 2), T7.51 (SEQ ID NO: 3), and SPT3 promoter (SEQ ID NO: 4), which are mutant promoters of the T7 promoter with attenuated transcriptional activity.

[0024] A specific example of a combination of a first promoter and a second promoter is a combination in which the first promoter is a T7 promoter and the second promoter is a promoter containing a base sequence selected from the base sequence shown in SEQ ID NO: 2 and the base sequence shown in SEQ ID NO: 3. The base sequence shown in SEQ ID NO: 2 (T7.5 promoter) and the base sequence shown in SEQ ID NO: 3 (T7.51 promoter) are promoters whose transcriptional activity has been weakened by introducing a mutation into the wild-type T7 promoter (SEQ ID NO: 1).

[0025] (Third and subsequent expression cassettes) The recombinant cell of this embodiment may further comprise one or more expression cassettes (a third expression cassette, a fourth expression cassette, ... an Nth expression cassette, ...) in addition to the first and second expression cassettes.

[0026] The Nth expression cassette comprises at least an Nth promoter and a polynucleotide sequence encoding a recombinant protein operably linked to the Nth promoter. The recombinant protein expressed by the Nth expression cassette is preferably the same as the recombinant protein expressed by the first expression cassette. The Nth expression cassette may further comprise one or more regulatory sequences operably linked to the Nth promoter and the polynucleotide sequence encoding the recombinant protein. The regulatory sequences are as described above.

[0027] The Nth promoter may be different from the first promoter and the second promoter, or may be the same as the first promoter or the second promoter. However, it is preferable that the Nth promoter be the same as a promoter with weaker transcriptional activity (e.g., the second promoter). This will make the effects of the present invention more pronounced. For example, when a recombinant cell has first, second, and third expression cassettes, it is preferable that the transcriptional activity of the second promoter is weaker than that of the first promoter, and that the third promoter is the same as the second promoter.

[0028] (recombinant protein) The recombinant protein (hereinafter also referred to as "target protein") produced by the recombinant cell of the present invention is not particularly limited, and any protein can be used. Examples of target proteins include any protein that is preferably produced on an industrial scale, such as proteins that can be used for industrial or medical purposes, and structural proteins. Specific examples of proteins that can be used for industrial or medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used in vaccinations, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, and full-length antibodies or antibody fragments or derivatives. Specific examples of structural proteins include fibroin (e.g., spider silk, silkworm silk, etc.), keratin, collagen, elastin, resilin, and fragments of these proteins, as well as proteins derived therefrom.

[0029] As used herein, fibroin includes naturally occurring fibroin and modified fibroin. As used herein, "naturally occurring fibroin" refers to fibroin having the same amino acid sequence as naturally occurring fibroin, and "modified fibroin" refers to fibroin having an amino acid sequence different from that of naturally occurring fibroin.

[0030] The fibroin may be spider silk fibroin. "Spider silk fibroin" includes natural spider silk fibroin and modified fibroins derived from natural spider silk fibroin. Examples of natural spider silk fibroin include spider silk proteins (SSPs) produced by spiders.

[0031] Fibroin can be, for example, a polymer represented by the formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) nThe fibroin according to this embodiment may be a protein containing a domain sequence represented by a motif. The fibroin according to this embodiment may further have amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeats of the amino acid motif characteristic of fibroin and consist of about 100 amino acid residues.

[0032] As used herein, the term "domain sequence" refers to a crystalline region specific to fibroin (typically, the amino acid sequence (A) n It is an amino acid sequence that generates a region (corresponding to a motif) and an amorphous region (typically corresponding to an REP in an amino acid sequence), and is represented by formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n The amino acid sequence represented by the motif (A) n The motif shows an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues is 2 to 27. (A) n The number of amino acid residues in the motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the motif is composed of only alanine residues). n At least seven of the motifs may be composed of only alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may also be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. (A) nThe motifs may have the same or different amino acid sequences, and the REPs present in multiple instances may have the same or different amino acid sequences.

[0033] Examples of naturally occurring fibroin include those represented by formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n Examples of naturally occurring fibroins include proteins containing domain sequences represented by motifs. Specific examples of naturally occurring fibroins include fibroins produced by insects or arachnids.

[0034] Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Antheraea pernyi, Eriogyna pyretorum, Pilosamia Cynthia ricini, Samia cynthia, Caligura japonica, Antheraea mylitta, and Antheraea assama, as well as hornet silk proteins excreted by larvae of the Japanese hornet (Vespa simillima xanthoptera).

[0035] A more specific example of fibroin produced by insects is silkworm fibroin L chain (GenBank accession numbers M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

[0036] Examples of fibroin produced by spiders include spiders belonging to the Araneus genus, such as the orb spider, the garden spider, the red orb spider, the green orb spider, and the bean spider; spiders belonging to the Neoscona genus, such as the mountain orb spider, the house spider, the dun orb spider, and the Satsuma spider; spiders belonging to the Pronus genus, such as the little orb spider; spiders belonging to the Cyrtarachne genus, such as the Japanese orb spider and the large orb spider; spiders of the genus Gasteracantha such as the Japanese bush spider and the Japanese bush spider; spiders of the genus Ordgarius such as the Japanese bush spider and the Japanese bush spider; spiders of the genus Argiope such as the orb-weaver spider, the orb-weaver spider and the long-jawed orb-weaver spider; spiders of the genus Arachnura such as the Japanese bush spider; spiders of the genus Acusilas such as the scraping spider; spiders of the genus C such as the orb-weaver spider, the orb-weaver spider and the Japanese bush spider; spider silk proteins produced by spiders belonging to the genus Poltys (such as the house spider), spiders belonging to the genus Cyclosa (such as the house spider, the four-headed house spider, the common house spider, and the black house spider), and spiders belonging to the genus Chorizopes (such as the Japanese canary spider), as well as spiders belonging to the genus Tetragnatha (such as the long-legged spider, the long-legged spider, the common house spider, and the scaly house spider), spiders belonging to the genus Tetragnatha (such as the long-legged spider, the common house spider, the large-legged spider), and ... spiders belonging to the genus Leucauge such as orb spiders and white orb spiders, spiders belonging to the genus Nephila such as orb spiders and giant orb spiders, spiders belonging to the genus Menosira such as golden spiders, spiders belonging to the genus Dyschiriognatha such as small red widow spiders, spiders belonging to the genus Latrodectus such as black widow spiders, redback spiders, gray widow spiders and three-spotted widow spiders,Examples of spider silk proteins include spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

[0037] An example of a protein derived from keratin is type I keratin from Capra hircus.

[0038] Examples of collagen-derived proteins include those represented by formula 3: [REP2] p (wherein, in formula 3, p represents an integer of 5 to 300. REP2 represents an amino acid sequence composed of Gly-XY, where X and Y represent any amino acid residues other than Gly. Multiple REP2s may have the same amino acid sequence or different amino acid sequences.)

[0039] Examples of proteins derived from elastin include proteins having amino acid sequences such as those under NCBI GenBank accession numbers AAC98395 (human), I47076 (ovine), and NP786966 (bovine).

[0040] Examples of proteins derived from resilin include those of formula 4: [REP3] q(wherein in formula 4, q is an integer of 4 to 300. REP3 is an amino acid sequence consisting of Ser-JJ-Tyr-Gly-U-Pro. J is any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. U is any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser. Multiple REP4s may have the same amino acid sequence or different amino acid sequences.)

[0041] The target protein may be either a hydrophilic or hydrophobic protein. The target protein is preferably one whose average HI, calculated by summing the hydrophobicity indices (HI) of all amino acid residues constituting the protein and then dividing the sum by the total number of amino acid residues (hereinafter referred to as "hydrophobicity"), is -1.0 or greater. The hydrophobicity index of amino acid residues is determined using a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein," J. Mol. Biol., 157, pp. 105-132). Specifically, the hydrophobicity index of each amino acid is as shown in Table 1 below.

[0042] [Table 1]

[0043] In one embodiment of the present invention, the hydrophobicity of the target protein may be -0.9 or greater, -0.8 or greater, -0.7 or greater, -0.6 or greater, -0.5 or greater, -0.4 or greater, -0.3 or greater, -0.2 or greater, -0.1 or greater, 0 or greater, 0.1 or greater, 0.2 or greater, 0.3 or greater, or 0.4 or greater; or the hydrophobicity of the target protein may be 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less.

[0044] The molecular weight of the target protein is not particularly limited, and may be, for example, 10 kDa to 700 kDa, or may be, for example, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, or may be, for example, 600 kDa or less, 500 kDa or less, 400 kDa or less, 300 kDa or less, or 200 kDa or less.

[0045] (Production of recombinant cells) A recombinant cell according to one embodiment of the present invention can be obtained, for example, by introducing a first expression cassette and a second expression cassette (and, if necessary, a third expression cassette, a fourth expression cassette, etc.) (hereinafter collectively referred to as "expression cassettes") into a host cell.

[0046] The recombinant cell according to this embodiment can be obtained, for example, by transforming a host cell with an expression vector having an expression cassette. The recombinant cell according to this embodiment may have the expression cassette outside the genomic DNA, or may have the expression cassette integrated into the genomic DNA, but is preferably one in which the expression cassette is integrated into the genomic DNA.

[0047] Known methods can be used to transform host cells, and examples include transforming host cells with a plasmid vector.

[0048] Methods for incorporating an expression cassette into genomic DNA can be known, including the λred method, which utilizes the recombination mechanism of λ phage double-strand break repair; Red / ET homologous recombination; and transposition using pUT-mini Tn5, which utilizes transposon activity. For example, an expression cassette can be incorporated into the genomic DNA of a host cell using Biomedal's "Transposon-Mediated Gene Transfer Kit: pUTmini-Tn5 Kit" according to the method described in the kit. In this case, the expression cassette may be incorporated into the genomic DNA of a host cell by recombining a DNA fragment containing at least a nucleic acid sequence encoding a target protein so that it is operably linked to one or more regulatory sequences in the genomic DNA of the host cell.

[0049] Preferred methods for transforming host cells include integrating an expression cassette into the genomic DNA of the host cell using λ phage integrase via an attachment site (attB site) in the genomic DNA of the host cell and an attachment site (attP site) on the vector, and integrating an expression cassette into the genomic DNA of the host cell using the Red-recombinase system, which uses the helper plasmid pKD46, which contains three genes essential for homologous recombination: exo, bet, and gamma genes.

[0050] As the host cells, any of prokaryotic cells such as bacteria, and eukaryotic cells such as yeast cells, filamentous fungal cells, insect cells, animal cells, and plant cells can be used. However, from the viewpoints of rapid growth and reduced culture costs, prokaryotic cells such as bacteria are preferred as host cells.

[0051] Prokaryotic host cells such as bacteria include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, and Pseudomonas. Preferred examples of prokaryotes include Escherichia coli, Bacillus subtilis, Pseudomonas, Corynebacterium, and Lactococcus. The host cell is preferably a microorganism belonging to the genus Escherichia, particularly Escherichia coli.

[0052] Examples of microorganisms belonging to the genus Escherichia include Escherichia coli BL21 (Novagen), Escherichia coli BL21(DE3) (Life Technologies), Escherichia coli BLR(DE3) (Merck Millipore), Escherichia coli DH1, Escherichia coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC 23506), Escherichia coli KY3276, Escherichia coli MC1000, Escherichia coli MG1655 (ATCC 47076), Escherichia coli No. 49, Escherichia coli Rosetta(DE3) (Novagen), Escherichia coli TB1, and Escherichia coli. Examples of suitable host cells include Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 (ATCC 27325), Escherichia coli XL1-Blue, and Escherichia coli XL2-Blue. The host cell is preferably Escherichia coli.

[0053] Any method for transforming the host cells can be used as long as it is a method for introducing DNA into the host cells, such as a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (Japanese Patent Laid-Open Publication No. 63-248394), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979).

[0054] Transformation of microorganisms belonging to the genus Brevibacillus can be carried out, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156:1130-1134), the method of Takagi et al. (Agric. Biol. Chem., 1989, 53:3099-3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61:202-203).

[0055] The type of vector used for transformation (hereinafter simply referred to as "vector") can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector. Examples of vectors include pBTrp2, pBTac1, and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (Japanese Patent Laid-Open Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (Stratagene), and pTrs30 [Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 (prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)), pGHA2 (prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091), pGKA2 (prepared from Escherichia coli IGKA2 (FERM Examples of suitable vectors include pGEX (prepared from U.S. Pat. Nos. 4,686,191, 4,939,094, and 5,160,735), pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094, and 5,160,735), pSupex, pUB110, pTP5, pC194, and pEG400 (J. Bacteriol., 172, 2392 (1990)), pGEX (Pharmacia), and the pET system (Novagen).

[0056] When Escherichia coli is used as the host cell, suitable vectors include pUC18, pBluescriptII, pSupex, pET22b, and pCold.

[0057] Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (Japanese Patent Laid-Open No. 2-31682), pNY700 (Japanese Patent Laid-Open No. 4-278091), pHY4831 (J. Bacteriol., 1987, pp. 1239-1245), pNU200 (Udaka Shigezo, Journal of the Japanese Society of Agricultural Chemistry, 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211 (J. Biochem., 1992, 112: 488-491), pNU2 Examples of such vectors include 11R2L5 (Japanese Patent Laid-Open No. 7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58:525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177:745-749), pHT210 (Japanese Patent Laid-Open No. 6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42:358-363), and pNCO2 (Japanese Patent Laid-Open No. 2002-238569), which is a shuttle vector between Escherichia coli and microorganisms belonging to the genus Brevibacillus.

[0058] The promoter is not limited as long as it satisfies the above-mentioned conditions and functions in the host cell. Examples include promoters derived from Escherichia coli or phages, such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. Artificially designed and modified promoters such as a promoter with two Ptrp promoters in tandem (Ptrp x 2), the tac promoter, the lacT7 promoter, and the letI promoter can also be used.

[0059] It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (a ribosome binding sequence) and the initiation codon is adjusted to an appropriate distance (e.g., 6 to 18 bases). A transcription termination sequence is not necessarily required, but it is preferable to place a transcription termination sequence immediately downstream of the gene encoding the target protein.

[0060] Eukaryotic host cells include, for example, yeast and filamentous fungi (molds, etc.).

[0061] Examples of yeast include yeasts belonging to the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, Yarrowia, and Hansenula.

[0062] When yeast is used as a host cell, the vector usually preferably contains an origin of replication (if amplification in the host cell is required) and a selectable marker for propagation of the vector in E. coli, an inducible promoter and terminator for recombinant protein expression in yeast, and a selectable marker for yeast.

[0063] If the vector is a non-integrative vector, it preferably further contains an autonomously replicating sequence (ARS), which can improve the stability of the vector in cells (Myers, AM, et al. (1986) Gene 45:299-310).

[0064] Examples of vectors used when yeast is used as a host cell include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, and pPICZ B.

[0065] When yeast is used as a host cell, the promoter is not particularly limited as long as it satisfies the above-mentioned conditions. Specific examples of promoters include the galactose-inducible gal1 promoter and gal10 promoter, the copper-inducible CUP1 promoter, the thiamine-inducible nmt1 promoter, and the methanol-inducible AOX1 promoter, AOX2 promoter, DHAS promoter, DAS promoter, FDH promoter, FMDH promoter, MOX promoter, ZZA1, PEX5-, PEX8-, and PEX14-promoters.

[0066] Any method for introducing a vector into yeast can be used as long as it is a method for introducing DNA into yeast, and examples thereof include the electroporation method (Methods Enzymol., 194, 182 (1990)), the spheroplast method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), the lithium acetate method (J. Bacteriol., 153, 163 (1983)), and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

[0067] Examples of filamentous fungi include fungi belonging to the genera Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, Penicillium, Myceliophtora, Botryts, Magnaporthe, Mucor, Metarhizium, Monascus, Rhizopus, and Rhizomucor.

[0068] When a filamentous fungus is used as a host cell, the promoter is not particularly limited as long as it satisfies the above-mentioned conditions. Specific examples of promoters include the salicylic acid-inducible PR1a promoter, the cycloheximide-inducible Placc promoter, and the quinic acid-inducible Pqa-2 promoter.

[0069] Vectors can be introduced into filamentous fungi using conventionally known methods, such as the method of Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)], the protoplast method [Mol. Gen. Genet., 168:111 (1979)], the competent method [J. Mol. Biol., 56:209 (1971)], and electroporation.

[0070] [Method for producing recombinant proteins] The method for producing a recombinant protein according to this embodiment includes at least a production step. The production step is a step of culturing the recombinant cell according to the present invention in a protein production medium. The method for producing a recombinant protein according to this embodiment may further include a pre-culture step of culturing the recombinant cell in a pre-culture medium prior to the production step.

[0071] The protein production medium for culturing recombinant cells is not particularly limited and can be selected from known natural or synthetic media depending on the type of recombinant cell. Examples of protein production media that can be used include liquid media containing components selected from carbon sources, nitrogen sources, phosphate sources, sulfur sources, vitamins, minerals, nutrients required for auxotrophy, and various other organic and inorganic components as needed. The types and concentrations of medium components can be determined appropriately by those skilled in the art.

[0072] The protein production medium preferably contains naturally occurring components. Naturally occurring components refer to components such as natural products (e.g., yeast) themselves, extracts from natural products (e.g., yeast extract), etc. The types and amounts of components contained in naturally occurring components are usually not completely specified. The naturally occurring components include, for example, at least one selected from the group consisting of vitamins, low molecular weight peptides (e.g., peptides with 2 to 20 amino acid residues), and amino acids.

[0073] Examples of carbon sources include sugars such as glucose, sucrose, lactose, galactose, fructose, and starch hydrolysates; alcohols such as glycerol and sorbitol; and organic acids such as fumaric acid, citric acid, and succinic acid.

[0074] The carbon source may be one type, or two or more types may be mixed at any ratio. The concentration of the carbon source in the protein production medium may be about 0.1 w / v% to 50 w / v%, preferably about 0.5 w / v% to 40 w / v%, more preferably about 1 w / v% to 30 w / v%, and particularly preferably about 5 w / v% to 20 w / v%. In this embodiment, glycerol or glucose is preferably used as the carbon source, and glycerol or glucose may be mixed with other carbon sources at any ratio. The ratio of glycerol or glucose in the carbon source is preferably 10 wt% or more, more preferably 50 wt% or more, and particularly preferably 70 wt% or more. The preferred initial concentration of the carbon source at the start of culture is as described above, but the carbon source may be added appropriately depending on the consumption of the carbon source during culture.

[0075] Nitrogen sources include inorganic nitrogen salts such as nitrates, ammonium salts, ammonia gas, and aqueous ammonia, as well as organic nitrogen sources such as amino acids, peptones, extracts, and corn steep liquor (CSL), a by-product of the cornstarch manufacturing industry. Peptones include casein peptone, meat peptone, myocardial peptone, gelatin peptone, and soybean peptone. Extracts include meat extract, yeast extract, and heart infusion. Nitrogen sources containing amino acids or peptides preferably contain a high content of lower molecular weight peptides and amino acids.

[0076] Examples of phosphate sources include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate.

[0077] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.

[0078] Vitamins include biotin, choline chloride, cyanocobalamin, folic acid, inositol, nicotinic acid, 4-aminobenzoic acid, pantothenic acid, pyridoxine, riboflavin, thianmine, thymidine, etc. Sources of vitamins include various extracts such as malt extract, potato extract, and tomato juice.

[0079] Minerals include phosphorus (P), sulfur (S), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), sodium (Na), etc.

[0080] The culture in the production step can be carried out aerobically, for example, by aeration culture or shaking culture. The culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Since the recombinant cell of the present invention can maintain the ability to produce a recombinant protein for a long period of time, the culture in the production step is preferably continuous culture or fed-batch culture.

[0081] The pH of the protein production medium may be, for example, 3.0 to 9.0, the culture temperature may be, for example, 15 to 40° C., and the culture time may be, for example, 1 to 60 hours.

[0082] The culture conditions are not particularly limited as long as they allow the recombinant cells to grow and allow the target protein to accumulate in the recombinant cells expressing the target protein. During the period in which the target protein is expressed, the recombinant cells may or may not grow. The culture conditions may or may not be the same before and after the initiation of target protein expression.

[0083] Culture temperature typically has a significant impact on cell growth. Generally speaking, the lower limit of growth is 0°C or slightly lower, which is the freezing point of water in cells, while the upper limit is determined by the denaturation temperature of macromolecular compounds such as proteins and nucleic acids. The temperature range in which a given strain can grow is relatively narrow. For example, the lower limit of growth for Escherichia coli is 0–15°C, the upper limit is 46°C, and the optimum temperature for growth is approximately 36–42°C. Microorganisms can be classified based on their optimum growth temperature: psychrophiles with an optimum temperature below 20°C, mesophiles with an optimum temperature between 20–45°C, and thermophiles with an optimum temperature above 45°C. Here, the optimum growth temperature refers to the temperature at which the cultured microorganism achieves its maximum specific growth rate. The specific growth rate refers to the growth rate per unit of microbial mass; this value is specific to each microorganism and varies depending on the culture conditions.

[0084] In one embodiment of the present invention, the "optimal growth temperature" refers to the temperature at which a microorganism can achieve the maximum specific growth rate when conditions other than the culture temperature, such as pH and dissolved oxygen concentration, are constant at the start of culture. In one embodiment of the present invention, while a recombinant cell is expressing a target protein (or after induction of expression if the expression of the target protein is inducible), the expression level of the target protein in the recombinant cell can be increased by cooling or maintaining the recombinant cell at a temperature lower than the optimal growth temperature for the recombinant cell, for example, by adjusting the culture temperature. A temperature lower than the optimal growth temperature for the recombinant cell may be, for example, 3 to 25°C lower, 8 to 20°C lower, 10 to 18°C ​​lower, 12 to 18°C ​​lower, 14 to 17°C lower, 3 to 10°C lower, or 5 to 8°C lower than the lower limit of the optimal growth temperature for the recombinant cell.

[0085] (Induction of recombinant protein expression) The recombinant cell according to this embodiment may be one in which expression of a recombinant protein (target protein) can be induced. Expression of the recombinant protein is induced by activating transcription (transcription of a nucleic acid encoding the target protein) by an inducible promoter. Activation of the inducible promoter can be performed according to methods known in the art, depending on the type of inducible promoter.

[0086] For example, when an inducible promoter that is activated in the presence of an inducer (expression inducer) such as isopropyl-β-thiogalactopyranoside (IPTG) is used, recombinant protein expression can be induced by adding the inducer to the culture medium. The inducer may be added to the culture medium all at once or in multiple batches, or may be added to the culture medium by continuous feeding. The inducer may also be added to the fed-batch substrate solution. The amount of inducer to be added can be determined depending on the type of inducer and inducible promoter, but can be, for example, in the range of 0.1 to 30 μg per gram of dry weight of recombinant cells, preferably 0.5 to 20 μg.

[0087] Furthermore, for example, when an inducible promoter that is activated by an increase or decrease in temperature is used, recombinant protein expression can be induced by increasing or decreasing the temperature of the culture medium. For example, when a λ phage PR promoter or PL promoter that is activated by an increase in temperature is used, recombinant protein expression during growth can be suppressed by maintaining the culture medium temperature in the range of 20 to 37°C during growth, and then recombinant protein expression can be induced by raising the culture medium temperature to 38 to 44°C. In order to mitigate the effects of heat shock proteins, as described in Japanese Patent Laid-Open No. 6-292563, the pH of the culture medium during growth can be set to 6.5 to 7.5, and then the pH of the culture medium can be changed to 4.5 to 6.5 at the time of starting induction of recombinant protein expression, thereby enabling more stable expression induction.

[0088] There are no particular limitations on the timing of transition from the stage of growing recombinant cells to the stage of inducing recombinant protein expression, and this can be set appropriately depending on the configuration of the culture system and the design of the production process. From the perspective of efficient recombinant protein production, it is preferable to start inducing recombinant protein expression when recombinant cell growth reaches the mid- to late-logarithmic growth phase.

[0089] The growth of recombinant cells begins with a lag or induction phase (a period in which the cell number increases slowly in the early stages of culture), passes through a logarithmic growth phase (a period in which the cell number doubles logarithmically per unit time), and reaches a stationary phase (a period in which no change is observed in the net number of cells). The mid-logarithmic growth phase refers to the period in which the cell number is intermediate between the number of cells in the lag phase and the number of cells in the stationary phase, and the late logarithmic growth phase refers to the period from the mid-logarithmic growth phase to the stationary phase. A specific example of the time to start inducing the expression of a recombinant protein is, for example, the OD 600 For recombinant cells, the OD is approximately 150. 600 It is preferable that the value of the saturation level is 30 to 110, more preferably 40 to 90, and even more preferably 50 to 80.

[0090] The time for inducing recombinant protein expression may be determined depending on the host used and the type of target protein, as long as a set production amount is reached. Because the production rate varies depending on culture conditions such as the temperature of the culture medium, it is not necessary to uniquely determine the time for inducing recombinant protein expression. The time for inducing recombinant protein expression may be set in accordance with the progress of the subsequent process of recombinant protein isolation and purification. In addition, in industrial production, it is preferable to set the time for inducing recombinant protein expression so as not to affect the parallel growth of recombinant cells and the transportation of the grown recombinant cells. Since the recombinant cells of the present invention can maintain their recombinant protein production ability for a long period of time, the longer the time for inducing recombinant protein expression, the more advantageous it is. Therefore, in one embodiment, but not limited to, the production process may involve adding an expression inducer to a protein production medium to induce recombinant protein expression, followed by culturing for 9 hours or more, 12 hours or more, or 15 hours or more.

[0091] (Pre-culture step) The pre-culture step is a step of culturing recombinant cells in a pre-culture medium prior to the production step. Specific aspects of the pre-culture medium are the same as those described above for the protein production medium. The pre-culture medium may be the same as or different from the protein production medium.

[0092] Expression constructs The expression construct of this embodiment has a first expression cassette comprising a polynucleotide sequence encoding a recombinant protein operably linked to a first promoter, and a second expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a second promoter different from the first promoter.

[0093] As used herein, "expression construct" refers to a nucleic acid that enables the expression of a target protein (recombinant protein). The expression construct according to this embodiment may be, for example, in the form of linear DNA having the minimum necessary components, or in the form in which the minimum necessary components are incorporated into a vector or the like. Specific aspects of the expression construct according to this embodiment are the same as those described for recombinant cells.

[0094] The expression construct according to this embodiment may be introduced into a host cell to express a target protein in vivo, or may be a cell-free construct to express a target protein in vitro.

[0095] When the expression construct according to this embodiment is used to express a target protein in vitro in a cell-free environment, the expression construct according to this embodiment may be provided as a kit containing the expression construct according to this embodiment and various components typically included in a cell-free protein synthesis kit. Examples of the various components typically included in a cell-free protein synthesis kit include cell extracts, substrates and / or energy sources for target protein synthesis, RNA polymerase, polyamines, salts, and oxidation / reduction regulators. These components may be premixed or individually included in the kit. [Example]

[0096] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0097] (1) Construction of mutant T7 promoter Mutations were introduced into the T7 promoter using genetic engineering techniques to obtain mutant promoters (T7.5 promoter: SEQ ID NO: 2, and T7.51 promoter: SEQ ID NO: 3) with weaker transcription activity than the T7 promoter.

[0098] The transcriptional activity of the T7 promoter, T7.5 promoter, T7.51 promoter, and T3 promoter was measured by quantitative PCR. Specifically, transcriptional activity was measured using ReverTra Ace (registered trademark) (TRT-101 / TOYOBO) according to the accompanying manual. The set of GizaQ Cterm_F01 (SEQ ID NO: 7) and GizaQ Cterm_R01 (SEQ ID NO: 8), which correspond to a partial sequence of the nucleotide sequence encoding the target protein (PRT799), was used as the primers for detecting transcriptional activity. The set of rrsA F (SEQ ID NO: 9) and rrsA R (SEQ ID NO: 10), which correspond to a partial sequence of a constantly expressed gene, was used as the control primers.

[0099] Figure 1 is a graph showing the results of quantitative PCR assays of the transcriptional activity of the T7 promoter (SEQ ID NO: 1), T7.5 promoter (SEQ ID NO: 2), T7.51 promoter (SEQ ID NO: 3), and SPT3 promoter (SEQ ID NO: 4). The vertical axis of the graph represents the ratio of the transcriptional activity of the target protein gene to the constitutively expressed gene (this ratio was calculated using the transcriptional activity value Cq1 of the polynucleotide sequence encoding the target protein and the transcriptional activity value Cqr of the polynucleotide sequence encoding the ribosome, a constitutively expressed gene, using the formula 2^(Cqr-Cq1)). The horizontal axis of the graph represents the time after induction of expression. As shown in Figure 1, the T7.5 promoter had weaker transcriptional activity than the T7 promoter and could not maintain its transcriptional activity for long periods of time. Furthermore, the T7.51 promoter had significantly weaker transcriptional activity than the T7 promoter, with almost no detectable transcriptional activity.

[0100] (2) Preparation of recombinant cells (E. coli strains expressing modified fibroin) (target protein) Based on the nucleotide and amino acid sequences of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), a modified fibroin (hereinafter also referred to as "PRT799") having the amino acid sequence shown in SEQ ID NO: 5 was designed. The amino acid sequence shown in SEQ ID NO: 5 has amino acid substitutions, insertions, and deletions of amino acid residues compared to the amino acid sequence of fibroin derived from Nephila clavipes to improve productivity, and further contains the amino acid sequences shown in SEQ ID NO: 6 (tag sequence and hinge sequence) added to the N-terminus.

[0101] Next, a nucleic acid encoding PRT799 was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. This nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised by restriction enzyme digestion with NdeI and EcoRI, and then recombined into the pET-22b(+) vector to obtain the pET-22(+) / PRT799 vector.

[0102] (Integration of the modified fibroin expression cassette into the host chromosome) Using Escherichia coli BL21(DE3) strain as the host, the modified fibroin expression cassette was integrated into three locations on the chromosome using the following methods (a) to (c), and recombinant cells carrying three modified fibroin expression cassettes were obtained.

[0103] (a)attHK022 The first modified fibroin expression cassette was integrated into the host chromosome by utilizing the lysogeny mechanism of the HK022 phage, which involves sequence-specific recombination between a specific site on the host chromosome (attB site) and a specific site on the phage genome (attP(HK022) site).

[0104] Figure 2 is a schematic diagram showing an outline of a method for integrating a modified fibroin expression cassette into a host chromosome using the lysogeny mechanism of HK022 phage. First, the nucleic acid encoding PRT799 was excised from the pET-22(+) / PRT799 vector by restriction enzyme digestion with NdeI and EcoRI, and then recombined with the attP(HK022) site-containing plasmid vector attHK022-Cm2-T7 (T7 promoter) or attHK022-Cm2-T7.51 (T7.51 promoter), to obtain the attHK022-T7p-PRT799-T7t-FRT-Cm2-ori_R6K-FRT vector or the attHK022-T7.51p-PRT799-T7t-FRT-Cm2-ori_R6K-FRT vector. Next, the attHK022-T7p-PRT799-T7t-FRT-Cm2-ori_R6K-FRT vector or the attHK022-T7.51p-PRT799-T7t-FRT-Cm2-ori_R6K-FRT vector was introduced into the host, and the modified fibroin (PRT799) expression cassette was integrated into the host chromosome by sequence-specific recombination between the attB site on the host chromosome and the attP(HK022) site of the same vector. The host was previously introduced with the helper plasmid pAH69 (J. Bact 183:6384-6393) containing the int gene to express integrase. Thereafter, the helper plasmid pCP20 (Proc. Natl. Acad. Sci. USA, 97: 6640-6645) was introduced to express FLP, thereby removing the chloramphenicol resistance gene flanked by FRT sequences and the ori_R6K region.

[0105] (b) attφ80 The second modified fibroin expression cassette was integrated into the host chromosome by utilizing the phage phage lysogenization mechanism, which involves sequence-specific recombination between a specific site on the host chromosome (attB site) and a specific site on the phage genome (attP site).

[0106] 3 is a schematic diagram showing an outline of a method for integrating a modified fibroin expression cassette into a host chromosome using the lysogenization mechanism of φ80 phage. First, the nucleic acid encoding PRT799 was excised from the pET-22(+) / PRT799 vector by restriction enzyme digestion with NdeI and EcoRI, and then recombined with the attP(φ80) site-containing plasmid vector attφ80-Km1_1-T7 (T7 promoter) or attφ80-Km1_1-T7.51 (T7.51 promoter) to obtain the attφ80-ori_R6K-FRT-Km1-FRT-T7p-PRT799-T7t-FRT vector or the attφ80-ori_R6K-FRT-Km1-FRT-T7.51p-PRT799-T7t-FRT vector. Next, the attφ80-ori_R6K-FRT-Km1-FRT-T7p-PRT799-T7t-FRT vector or the attφ80-ori_R6K-FRT-Km1-FRT-T7.51p-PRT799-T7t-FRT vector was introduced into the host into which the first modified fibroin expression cassette had been integrated by the method (a) above, and the second modified fibroin (PRT799) expression cassette was integrated into the host chromosome by sequence-specific recombination between the attB site on the host chromosome and the attP(φ80) site of the same vector. Subsequently, the kanamycin resistance gene flanked by the FRT sequences was removed by introducing the helper plasmid pCP20 to express FLP.

[0107] (c)λRed_manX The third modified fibroin expression cassette was integrated into the host chromosome using the homologous recombination system of λ phage, which causes homologous recombination with the exo, bet, and gam gene products in the Red region of the phage genome.

[0108] Figure 4 is a schematic diagram outlining a method for integrating a modified fibroin expression cassette into a host chromosome using the homologous recombination system of λ phage. First, a modified fibroin expression cassette (containing the manX5' homologous sequence, SPT3 promoter, PRT799, and T7 terminator, in this order) was amplified by PCR using primers that introduce modifications into the T7 promoter, with the pET-22(+) / PRT799 vector as a template. Similarly, a chloramphenicol resistance gene expression cassette (containing the T7 terminator homologous sequence, FRT, chloramphenicol resistance gene, and FRT-manX3' homologous sequence, in this order) was amplified by PCR using the pKD13-Cm vector as a template. Both PCR products were ligated using the In-Fusion® Cloning System (Takara Bio Inc.). Next, the linked DNA fragment was introduced into a host incorporating the first and second modified fibroin expression cassettes using methods (a) and (b) above. The third modified fibroin (PRT799) expression cassette was integrated into the host chromosome by homologous recombination between the manX5' homologous sequence on the host chromosome and the manX5' homologous sequence on the DNA fragment, and by homologous recombination between the manX3' homologous sequence on the host chromosome and the manX3' homologous sequence on the DNA fragment. The host was previously introduced with the helper plasmid pKD46 (Proc. Natl. Acad. Sci. USA, 97:6640-6645), which contains the exo, bet, and gam genes, to express each gene. The chloramphenicol resistance gene flanked by FRT sequences was then removed by introducing the helper plasmid pCP20 to express FLP.

[0109] (3) Expression and evaluation of modified fibroin The expression level of modified fibroin (PRT799) was evaluated using two types of recombinant cells obtained by the method (2) above. The two types of recombinant cells used were a recombinant cell in which all three modified fibroin expression cassettes had a T7 promoter (hereinafter also referred to as the "T7-T7-T7 strain"), and a recombinant cell in which one of the three modified fibroin expression cassettes had a T7 promoter and the remaining two had T7.51 promoters (hereinafter also referred to as the "T7-T7.51-T7.51 strain").

[0110] The T7-T7-T7 strain and the T7-T7.51-T7.51 strain were cultured in 2 mL of LB medium for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 2) at OD . 600 The culture temperature was kept at 30°C, and the OD 600 The flask culture was continued until the fertilization rate reached 5 (about 15 hours), and a seed culture solution was obtained. [Table 2]

[0111] The seed culture solution was added to a jar fermenter containing 500 mL of production medium (Table 3) and measured at OD 600 The culture temperature was maintained at 37°C and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration.

[0112] [Table 3]

[0113] Immediately after the glucose in the production medium was completely consumed, the feed solution (the fed-batch substrate solution in Table 4) was added at a rate of 6 g / hour. The culture temperature was maintained at 37°C, and the pH was controlled at a constant level of 6.9. The culture was continued for 16 hours, with the dissolved oxygen concentration in the culture maintained at 20% of the dissolved oxygen saturation concentration. 1 M isopropyl-β-thiogalactopyranoside (IPTG) was then added to the culture to a final concentration of 0.1 mM to induce expression of the modified fibroin. SDS-PAGE was performed using cells prepared from the culture medium before and after the addition of IPTG. Expression of the desired modified fibroin was confirmed by the appearance of a band of the desired modified fibroin size, which was dependent on the addition of IPTG.

[0114] [Table 4]

[0115] The collected cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF and disrupted using a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until highly purified. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL and dissolved by stirring at 60°C for 30 minutes. After dissolution, the suspension was dialyzed against water using a dialysis tube (Cellulose tube 36 / 32, Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was collected by centrifugation, and the water was removed using a freeze-dryer. The freeze-dried powder was collected to obtain modified fibroin (PRT799).

[0116] The resulting freeze-dried powder was subjected to polyacrylamide gel electrophoresis, and image analysis was performed using Totallab (Nonlinear Dynamics Ltd.) to evaluate the amount of modified fibroin produced. The amount of each modified fibroin produced (per cell) was calculated from the weight of the freeze-dried powder and expressed as a relative value, with the value after 15 hours of induction in the T7-T7-T7 strain set at 100%.

[0117] (4) Results Figure 5 is a graph showing the results of evaluating the protein (modified fibroin) production yields of the T7-T7-T7 strain and the T7-T7.51-T7.51 strain. As shown in Figure 5, the T7-T7.51-T7.51 strain, in which expression of the three modified fibroin genes is driven by the T7 promoter or the T7.51 promoter, respectively, produced a higher amount of protein (modified fibroin) than the T7-T7-T7 strain, in which expression of all three modified fibroin genes is driven by the T7 promoter. Furthermore, the difference in protein (modified fibroin) production yields increased with time after induction. This is thought to reflect the ability of the T7-T7.51-T7.51 strain to maintain its ability to produce recombinant proteins for a long period of time.

Claims

1. A recombinant cell comprising a first expression cassette comprising a polynucleotide sequence encoding a recombinant protein operably linked to a first promoter, and a second expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a second promoter different from the first promoter, wherein the transcriptional activity of the second promoter is 50 or less when the transcriptional activity of the first promoter is taken as 100.

2. 2. The recombinant cell of claim 1, wherein the polynucleotide sequences of the first promoter and the second promoter have at least 80% sequence identity.

3. 3. The recombinant cell of claim 1, wherein the first promoter is a T7 promoter.

4. The recombinant cell according to any one of claims 1 to 3, wherein the second promoter comprises a base sequence selected from the base sequence shown in SEQ ID NO: 2 and the base sequence shown in SEQ ID NO:

3.

5. The recombinant cell of any one of claims 1 to 4, further comprising a third expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a third promoter.

6. The recombinant cell of claim 5 , wherein the third promoter is the same as the first promoter or the second promoter.

7. The recombinant cell according to any one of claims 1 to 6, wherein the recombinant protein is fibroin.

8. The recombinant cell according to any one of claims 1 to 7, wherein the first expression cassette and the second expression cassette are integrated into host genomic DNA.

9. A method for producing a recombinant protein, comprising a production step of culturing in a protein production medium a recombinant cell having a first expression cassette comprising a polynucleotide sequence encoding a recombinant protein operably linked to a first promoter, and a second expression cassette comprising a polynucleotide sequence encoding the recombinant protein operably linked to a second promoter different from the first promoter, wherein the transcription activity of the second promoter is 50 or less when the transcription activity of the first promoter is taken as 100.

10. The method according to claim 9 , wherein the recombinant cells are cultured by continuous culture or fed-batch culture in the production step.

11. The production method according to claim 9 or 10, wherein the production process includes an expression induction step of inducing expression of the recombinant protein by adding an expression inducer to the protein production medium.

12. The production method according to any one of claims 9 to 11, wherein the production step comprises adding an expression inducer to the protein production medium to induce expression of the recombinant protein, and then culturing the recombinant protein for 9 hours or more.

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