JP7762935B2 - Recombinant cells, expression constructs, and methods for producing recombinant proteins - Google Patents

Description

The present invention relates to recombinant cells, expression constructs, and methods for producing recombinant proteins.

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 large-scale production of recombinant proteins.

To produce large quantities 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 to produce recombinant proteins, codon optimization of the polynucleotide sequence encoding the target protein has been performed. 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.

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 using methods such as homologous recombination (see, for example, Patent Document 1).

Patent No. 5996052

However, high expression of recombinant proteins from 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, it can cause a shortage of resources for protein expression (RNA and protein raw materials, the intracellular machinery for transcription and translation), increasing the metabolic burden on the host cell. As a result, recombinant protein yield is suppressed. That is, once the recombinant protein yield reaches a certain level, the recombinant protein production capacity gradually decreases. Therefore, maintaining sustainable production capacity 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).

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.

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]
[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 comprises adding an expression inducer to the protein production medium to induce expression of the recombinant protein, followed by culturing for 9 hours or more.

The present invention makes it possible to provide recombinant cells that express increased amounts of recombinant proteins. The present invention also makes it possible to provide expression constructs that express increased amounts of recombinant proteins.

1 is a graph showing the results of quantitative PCR measurement of the transcriptional activity of the T7 promoter, T7.5 promoter, T7.51 promoter, and SP T3 promoter. 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. 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. 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. 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.

The following describes in detail the embodiments of the present invention. However, the present invention is not limited to the following embodiments.

[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.

(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.

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).

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 promoter or an artificially produced promoter. In the present invention, promoters with at least two different transcriptional activities are used, and these promoters may be of different origins or may be of the same origin.

(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.

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 are as described above.

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

(First and second promoters)
The second promoter is preferably a promoter whose transcription activity is 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.

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.

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 produced, for example, by genetic engineering techniques.

In one embodiment of the present 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.

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.

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 sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. The base sequences shown in SEQ ID NO: 2 (T7.5 promoter) and 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).

(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.

The Nth expression cassette includes at least the 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 include 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.

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 even more pronounced. For example, if a recombinant cell has first, second, and third expression cassettes, it is preferable that the transcriptional activity of the second promoter be weaker than the transcriptional activity of the first promoter, and that the third promoter be the same as the second promoter.

(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.

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.

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.

Fibroin may be, for example, a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif. The fibroin according to this embodiment may have further 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 are not limited to, regions that do not have repetitions of the amino acid motif characteristic of fibroin and consist of about 100 amino acid residues.

As used herein, the term "domain sequence" refers to an amino acid sequence that generates a crystalline region (typically corresponding to the (A) _n motif in the amino acid sequence) and an amorphous region (typically corresponding to REP in the amino acid sequence) that are specific to fibroin, and refers to an amino acid sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif. Here, the (A) _n motif represents an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. The number of amino acid residues in the (A) _n 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. Furthermore, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) _n 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 only of alanine residues). At least seven of the (A) _n motifs present in multiple instances in the domain sequence may be composed only of 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. The multiple (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. The multiple REPs present may be the same amino acid sequence as each other or different amino acid sequences.

Examples of naturally occurring fibroins include proteins containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. Specific examples of naturally occurring fibroins include fibroins produced by insects or arachnids.

Fibroin produced by insects includes, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Pilosamia cynthia ricini, Samia cynthia, Caligrassworm, Antheraea mylitta, and Antheraea japonica. Examples include silk proteins produced by silkworms such as silkworms (Bombyx mori), and hornet silk proteins excreted by hornet larvae (Vespa simillima xanthoptera).

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)).

Fibroin produced by spiders includes, for example, 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 belonging to the Gaster genus, such as the spiny orb spider and the Japanese orb spider; spiders belonging to the genus Acantha, spiders belonging to the genus Ordgarius such as the orb-weaver spider and the black widow spider, spiders belonging to the genus Argiope such as the orb-weaver spider, spiders belonging to the genus Arachnura such as the brown recluse spider, spiders belonging to the genus Acusilas such as the scraping spider, spiders belonging to the genus Cytophora such as the orb-weaver spider, the orb-weaver spider and the black widow spider, spiders belonging to the genus Poltys such as the house spider spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the dung beetle, four-headed dung beetle, brown dung beetle, and black dung beetle, and spiders belonging to the genus Chorizopes, such as the Japanese canary spider; spiders belonging to the genus Tetragnatha, such as the long-legged spider, the long-legged spider, the broad-legged spider, and the scaly canary spider; spiders belonging to the genus Leucauge, such as the large white weaver spider, the medium-sized weaver spider, and the small white weaver spider; spider silk proteins produced by spiders belonging to the genus Orb spider, the giant orb spider, and the like. Examples of spider silk proteins include those produced by spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as the golden orb spider, spiders belonging to the genus Dyschiriognatha such as the small brown recluse spider, spiders belonging to the genus Latrodectus such as the black widow spider, redback spider, grey widow spider and ten-spotted latrodectus, and spiders belonging to the family Tetragnathiidae 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).

Examples of keratin-derived proteins include type I keratin from Capra hircus.

An example of a collagen-derived protein is a protein comprising a domain sequence 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-X-Y, 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).

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

An example of a protein derived from resilin is a protein comprising a domain sequence represented by 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-J-J-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).

The target protein may be either a hydrophilic or hydrophobic protein. The target protein is preferably one whose average HI (hereinafter also referred to as "hydrophobicity"), calculated by summing the hydrophobicity index (HI) of all amino acid residues constituting the target protein and then dividing this sum by the total number of amino acid residues, is -1.0 or greater. The hydrophobicity index of amino acid residues is determined using a known index (Hydrophobicity 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.

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. 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.

The molecular weight of the target protein is not particularly limited, and may be, for example, 10 kDa or more and 700 kDa or less. The molecular weight of the target protein 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, and 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.

(Preparation 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, ...) (hereinafter collectively referred to as "expression cassettes") into a host cell.

The recombinant cell of this embodiment can be obtained, for example, by transforming a host cell with an expression vector having an expression cassette. The recombinant cell of this embodiment may have the expression cassette outside of the genomic DNA, or may have the expression cassette integrated into the genomic DNA, but it is preferable that the expression cassette be integrated into the genomic DNA.

Known methods can be used to transform host cells, such as transforming host cells with a plasmid vector.

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

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

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

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. It is preferred that the host cell be a microorganism belonging to the genus Escherichia, particularly Escherichia coli.

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), and Escherichia coli No. Examples of suitable host cells include Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, 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.

The host cells can be transformed by any method that involves introducing DNA into the host cells. Examples include the calcium ion method [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP 63-248394 A), or the methods described in Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).

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).

The type of vector used for transformation (hereinafter simply referred to as "vector") can be selected appropriately depending on the type of host, such as a plasmid vector, viral vector, cosmid vector, fosmid vector, or 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 (JP 58-110600 A), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], and pLSA1 [Agric. Biol. Chem., 48, 669 (1984)]. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (Stratagene), pTrs30 [prepared from 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 pTerm2 (US Patent 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).

When E. coli is used as the host cell, suitable vectors include pUC18, pBluescript II, pSupex, pET22b, and pCold.

Specific examples of vectors suitable for microorganisms belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP Patent Publication No. 2-31682), pNY700 (JP Patent Publication 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), and pNU2 Examples of such vectors include 11R2L5 (JP 7-170984 A), pNH301 (Appl. Environ. Microbiol., 1992, 58:525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177:745-749), pHT210 (JP 6-133782 A), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42:358-363), and pNCO2 (JP 2002-238569 A), a shuttle vector between Escherichia coli and microorganisms belonging to the genus Brevibacillus.

There are no limitations on the promoter, so long as it meets the above-mentioned conditions and functions in the host cell. Examples include promoters derived from E. 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 let I promoter can also be used.

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

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

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

When yeast is used as a host cell, the vector typically 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.

If the vector is a non-integrating vector, it preferably further contains an autonomously replicating sequence (ARS). This can improve the stability of the vector within the cell (Myers, A.M., et al. (1986) Gene 45:299-310).

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

When yeast is used as a host cell, the promoter is not particularly limited as long as it meets the above-mentioned conditions. Specific examples of promoters include the galactose-inducible gal 1 promoter and gal 10 promoter; the copper-inducible CUP 1 promoter; the thiamine-inducible nmt 1 promoter; and the methanol-inducible AOX 1 promoter, AOX 2 promoter, DHAS promoter, DAS promoter, FDH promoter, FMDH promoter, MOX promoter, ZZA 1, PEX 5-, PEX 8-, and PEX 14-promoters.

Any method for introducing DNA into yeast can be used to introduce a vector into yeast, including, for example, electroporation (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).

Examples of filamentous fungi include the genus Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Humicola, and Penicillium. Examples of fungi that can be used include those belonging to the genera Illium, Myceliophtora, Botrytis, Magnaporthe, Mucor, Metarhizium, Monascus, Rhizopus, and Rhizomucor.

When using a filamentous fungus as a host cell, the promoter is not particularly limited as long as it meets 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.

Vectors can be introduced into filamentous fungi using conventional methods. Examples include 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.

[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.

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. For example, a liquid medium containing components selected from a carbon source, nitrogen source, phosphate source, sulfur source, vitamins, minerals, nutrients required for auxotrophy, and various other organic and inorganic components as needed can be used as the protein production medium. The types and concentrations of medium components can be determined appropriately by those skilled in the art.

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 naturally occurring components typically are not fully specified. 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.

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.

The carbon source may be a single type, or two or more types may be mixed in any ratio. The concentration of the carbon source in the protein production medium may be approximately 0.1 w/v% to 50 w/v%, preferably approximately 0.5 w/v% to 40 w/v%, more preferably approximately 1 w/v% to 30 w/v%, and particularly preferably approximately 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 in 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 cultivation is as described above, but the carbon source may be added appropriately depending on the consumption of the carbon source during cultivation.

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. Examples of peptones include casein peptone, meat peptone, myocardial peptone, gelatin peptone, and soybean peptone. Examples of 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.

Phosphate sources include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate.

Sulfur sources include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.

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

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

The culture in the production process 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 of these. Because the recombinant cells of the present invention can maintain the ability to produce recombinant proteins for long periods of time, the culture in the production process is preferably continuous culture or fed-batch culture.

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. The culture time may be, for example, 1 to 60 hours.

There are no particular limitations on the culture conditions, so 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 target protein begins to be expressed.

Cultivation 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 high-molecular-weight 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 around 36-42°C. Microorganisms can be classified by their optimum growth temperature into 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 microorganism being cultured achieves its maximum specific growth rate. Furthermore, the specific growth rate refers to the growth rate per unit of microbial mass; it is a value specific to each microorganism and varies depending on the culture conditions.

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 the recombinant cells are 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 cells can be increased by cooling or maintaining the recombinant cells at a temperature lower than the optimal growth temperature of the recombinant cells, for example, by adjusting the culture temperature. A temperature lower than the optimal growth temperature of the recombinant cells 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 of the recombinant cells.

(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.

For example, when using an inducible promoter that is activated in the presence of an inducer (expression inducer) such as isopropyl-β-thiogalactopyranoside (IPTG), 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 it 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 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.

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 using the λ phage PR promoter or PL promoter, which is activated by an increase in temperature, recombinant protein expression during growth can be suppressed by setting the culture medium temperature during growth in the range of 20-37°C, and then recombinant protein expression can be induced by raising the culture medium temperature to 38-44°C. In order to mitigate the effects of heat shock proteins, as described in JP 6-292563 A, the pH of the culture medium during growth can be set to 6.5-7.5, and then the pH of the culture medium can be changed to 4.5-6.5 at the time of starting the induction of recombinant protein expression, thereby enabling more stable expression induction.

There are no particular limitations on the timing of the 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 has reached the mid- to late-logarithmic growth phase.

The growth of recombinant cells begins with a lag phase 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 fluctuations in the net number of cells are observed). The mid-logarithmic growth phase refers to the period in which the cell number is intermediate between the lag phase and the stationary phase, and the late logarithmic growth phase refers to the period from the mid-logarithmic growth phase to the stationary phase. As a specific example of the time to start inducing recombinant protein expression, for example, in the case of recombinant cells whose _OD600 value in the stationary phase is approximately 150, it is preferably when the _OD600 value reaches 30 to 110, more preferably when it reaches 40 to 90, and even more preferably when it reaches 50 to 80.

The time for inducing recombinant protein expression may be determined depending on the host used and the type of target protein, and may be until a set production amount is reached. Because the production rate varies depending on culture conditions such as the temperature of the culture medium, there is no need 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 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 or the transportation of the grown recombinant cells. Since the recombinant cells of the present invention can maintain their recombinant protein production ability for an extended 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.

(Preculture 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.

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.

As used herein, the term "expression construct" refers to a nucleic acid that enables the expression of a target protein (recombinant protein). The expression construct of 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 of this embodiment are the same as those described for recombinant cells.

The expression construct of 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.

When the expression construct of this embodiment is used to express a target protein in vitro in a cell-free environment, the expression construct of this embodiment may be provided as a kit containing the expression construct of this embodiment and various components typically included in cell-free protein synthesis kits. Examples of the various components typically included in cell-free protein synthesis kits 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.

The present invention will be explained in more detail below based on examples. However, the present invention is not limited to the following examples.

(1) Preparation of mutant T7 promoters 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.

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 primers used to detect transcriptional activity were a 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), and the control primers used were a set of rrsA F (SEQ ID NO: 9) and rrsA R (SEQ ID NO: 10), which correspond to a partial sequence of a constitutively expressed gene.

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 formula 2^(Cqr-Cq1), where Cq1 is the transcriptional activity value of the polynucleotide sequence encoding the target protein and Cqr is the transcriptional activity value of the polynucleotide sequence encoding the ribosome, a constitutively expressed gene). 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 was unable to maintain transcriptional activity for long periods of time. Furthermore, the T7.51 promoter had significantly weaker transcriptional activity than the T7 promoter, with barely detectable transcriptional activity.

(2) Preparation of recombinant cells (E. coli strain expressing modified fibroin) (target protein)
A modified fibroin (hereinafter also referred to as "PRT799") having the amino acid sequence shown in SEQ ID NO: 5 was designed based on the nucleotide sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415). The amino acid sequence shown in SEQ ID NO: 5 has an amino acid sequence in which amino acid residues have been substituted, inserted, and deleted with the aim of improving productivity compared to the amino acid sequence of fibroin derived from Nephila clavipes, and further has the amino acid sequence shown in SEQ ID NO: 6 (tag sequence and hinge sequence) added to the N-terminus.

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 stop codon. This nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised using restriction enzymes NdeI and EcoRI, and then recombined into the pET-22b(+) vector to obtain the pET-22(+)/PRT799 vector.

(Integration of modified fibroin expression cassette into host chromosome)
Using Escherichia coli BL21 (DE3) strain as a host, modified fibroin expression cassettes were 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.

(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).

Figure 2 is a schematic diagram outlining a method for integrating a modified fibroin expression cassette into a host chromosome using the lysogenization mechanism of the HK022 phage. First, the nucleic acid encoding PRT799 was excised from the pET-22(+)/PRT799 vector by restriction enzyme digestion with NdeI and EcoRI. This was 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 a helper plasmid pAH69 (J. Bact 183: 6384-6393) containing the int gene to express integrase. The helper plasmid pCP20 (Proc. Natl. Acad. Sci. USA, 97: 6640-6645) was then introduced to express FLP, thereby removing the chloramphenicol resistance gene flanked by FRT sequences and the ori_R6K region.

(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).

Figure 3 is a schematic diagram outlining a method for integrating a modified fibroin expression cassette into a host chromosome using the φ80 phage lysogenization mechanism. First, the nucleic acid encoding PRT799 was excised from the pET-22(+)/PRT799 vector by restriction enzyme digestion with NdeI and EcoRI. This was 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), yielding 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 using 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. The kanamycin resistance gene flanked by FRT sequences was then removed by introducing the helper plasmid pCP20 and expressing FLP.

(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 via the exo, bet, and gam gene products in the Red region of the phage genome.

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, the 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, the chloramphenicol resistance gene expression cassette (containing the T7 terminator homologous sequence, FRT, chloramphenicol resistance gene, FRT, and 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, and 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) containing 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.

(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 in (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").

The T7-T7-T7 strain and the T7-T7.51-T7.51 strain were each cultured in 2 mL of LB medium for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 2) so that the OD ₆₀₀ became 0.005. The culture solution temperature was maintained at 30°C, and flask culture was continued until the OD ₆₀₀ reached 5 (approximately 15 hours), to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 3) so that the OD ₆₀₀ was 0.05. The culture temperature was maintained at 37°C, and the pH was controlled to a constant 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.

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 culture was controlled at a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 16 hours. 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 IPTG addition, and the appearance of a band of the desired modified fibroin size, which was dependent on IPTG addition, confirmed the expression of the desired modified fibroin.

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 the cells were 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 solution was dialyzed against water using a dialysis tube (Cellulose tube 36/32 manufactured by 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).

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%.

(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 was driven by the T7 promoter or the T7.51 promoter, respectively, had a higher protein (modified fibroin) production yield than the T7-T7-T7 strain, in which expression of all three modified fibroin genes was driven by the T7 promoter. Furthermore, the difference in protein (modified fibroin) production yields increased with the passage of 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 (12)

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. The recombinant cell of claim 1, wherein the polynucleotide sequences of the first promoter and the second promoter have at least 80% sequence identity. The recombinant cell of claim 1 or 2, wherein the first promoter is a T7 promoter. 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. The recombinant cell described in 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. The recombinant cell of claim 5, wherein the third promoter is the same as the first promoter or the second promoter. The recombinant cell described in any one of claims 1 to 6, wherein the recombinant protein is fibroin. The recombinant cell described in any one of claims 1 to 7, wherein the first expression cassette and the second expression cassette are integrated into host genomic DNA. A method for producing a recombinant protein, comprising a production step of culturing, in a protein production medium, recombinant cells having a first expression cassette containing a polynucleotide sequence encoding a 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, wherein the transcription activity of the second promoter is 50 or less when the transcription activity of the first promoter is taken as 100. The method according to claim 9 , wherein the recombinant cells are cultured by continuous culture or fed-batch culture in the production step. 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. 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, followed by culturing for 9 hours or more.