WO2021020546A1 - Cellule recombinée et procédé de production de protéine recombinée - Google Patents

Cellule recombinée et procédé de production de protéine recombinée Download PDF

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WO2021020546A1
WO2021020546A1 PCT/JP2020/029376 JP2020029376W WO2021020546A1 WO 2021020546 A1 WO2021020546 A1 WO 2021020546A1 JP 2020029376 W JP2020029376 W JP 2020029376W WO 2021020546 A1 WO2021020546 A1 WO 2021020546A1
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recombinant
protein
acid sequence
nucleic acid
sula
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Japanese (ja)
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昂文 野田
研二 津山
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Ｓｐｉｂｅｒ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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  • the present invention relates to a method for producing recombinant cells and recombinant proteins. More specifically, the present invention relates to a recombinant cell into which a nucleic acid sequence encoding sulA has been introduced to produce a recombinant protein, and a method for producing a recombinant protein using the recombinant cell.
  • Non-Patent Document 1 a recombinant protein expression system using mammalian cells, yeast, mold, gram-negative bacteria, gram-positive bacteria and the like as hosts is known, and these are also used for industrial production (for example,).
  • An object of the present invention is to provide a recombinant cell and a production method capable of efficiently producing a recombinant protein.
  • the present inventors use a cell as a host, which can produce the recombinant protein and can change the volume of the cell at an arbitrary culture time, so that the recombinant protein per cell can be produced. It was found that the production volume of The present invention is based on this novel finding.
  • the present invention relates to, for example, the following inventions.
  • a nucleic acid sequence containing a nucleic acid sequence encoding a recombinant protein and one or more regulatory sequences operably linked to the nucleic acid sequence and encoding the morphogenesis control factor sulA has been introduced.
  • Recombinant cells [2] The recombinant cell according to [1], wherein the nucleic acid sequence encoding sulA has at least 90% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 2.
  • recombinant cell according to any one of [1] to [8], wherein the recombinant cell is a microorganism belonging to the genus Escherichia.
  • a nucleic acid sequence containing a nucleic acid sequence encoding a recombinant protein and one or more regulatory sequences operably linked to the nucleic acid sequence and encoding a morphogenesis control factor sulA has been introduced.
  • a method for producing a recombinant protein which comprises a production step of culturing the recombinant cell in a protein production medium.
  • FIG. 5 is a schematic diagram showing an outline of a method for incorporating a modified fibroin expression cassette onto a host chromosome by utilizing the mechanism by which HK022 phages are lysogenized. It is a schematic diagram which shows the outline of the method of incorporating a modified fibroin expression cassette on a host chromosome by utilizing the homologous recombination system possessed by ⁇ 80 phage. It is a schematic diagram which shows the outline of the method of incorporating a modified fibroin expression cassette on a host chromosome by utilizing the mechanism of lysogenization of ⁇ phage.
  • the recombinant cell contains a nucleic acid sequence encoding a recombinant protein and one or more regulatory sequences operably linked to the nucleic acid sequence, and controls morphology.
  • a nucleic acid sequence encoding the factor sulA has been introduced.
  • the recombinant cell according to the present embodiment contains a nucleic acid sequence encoding a recombinant protein (target protein) and one or more regulatory sequences operably linked to the nucleic acid sequence, that is, expresses the target protein.
  • An expression cassette (hereinafter, also referred to as “target protein expression cassette”) is included.
  • the recombinant cell according to the present embodiment may contain one target protein expression cassette, or may contain a plurality of (for example, two, three, four, five).
  • a nucleic acid sequence encoding the morphogenesis control factor sulA that is, a cassette (hereinafter, also referred to as “sulA expression cassette”) for expressing sulA has been introduced.
  • the recombinant cell according to the present embodiment may contain one sulA expression cassette, or may contain a plurality of cells (for example, two, three, four, five).
  • one or both of the target protein expression cassette and the sulA expression cassette may be inducibly expressed, and for the induction of expression, for example, an inducible promoter can be used. That is, the target protein and / or sulA are regulated by an inducible promoter.
  • the protein of interest and the sulA expression cassette are preferably controlled by different inducible promoters.
  • Morphogenesis control factor sulA means a protein related to cell morphology control.
  • Cell morphology includes, for example, cell stiffness, shape, and volume.
  • Morphogenic regulators include, for example, proteins associated with the formation or regulation of cell elongation, cell width and cell polarity.
  • peptidoglycan saliva chains cross-linked by short peptides.
  • the degradation of pre-existing peptidoglycan and the insertion of newly synthesized peptidoglycan are strictly controlled during cell elongation and division to maintain the morphology of the cell without rupture. Is possible.
  • One of the functions of cytoskeletal proteins is thought to be to control the intracellular localization of peptidoglycan synthase.
  • peptidoglycan in the periplasmic region ultimately determines the morphology of the cell, but the enzyme that synthesizes it is controlled by the cytoskeletal protein in the cytoplasm and the protein that controls the function of the cytoskeletal protein. is there.
  • Bacterial cytoskeletal proteins include, for example, FtsZ tubulin and MreB actin.
  • the peptidoglycan synthase include PBP3 (FtsI) and PBP2.
  • FtsZ tubulin is the first of the mitotic related proteins to localize to the mitotic plane and form a mitotic ring (Z ring).
  • more than a dozen kinds of related proteins are assembled one after another in a Z ring to form a supramolecular complex called divisome with PBP3.
  • MreB forms a complex called elongasome with PBP2 and the like, which are essential for cell elongation.
  • constituent factors of elongasome include RodZ, RodA, MreC, and MreD.
  • Proteins that control the function of cytoskeletal proteins include, for example, in prokaryotes, sulA, yeV, slmA and Min family proteins (MinC, D, and E).
  • some microorganisms such as Bacillus include ezrA and Noc.
  • sulA is a component of the SOS system and inhibits the polymerization of FtsZ by interacting with FtsZ.
  • the wild-type sulA has the amino acid sequence shown in SEQ ID NO: 1, and the sulA gene encoding this has, for example, the nucleic acid sequence shown in SEQ ID NO: 2.
  • the nucleic acid sequence encoding the morphogenesis control factor sulA in this embodiment has at least 90%, preferably 93%, 95%, 98% or 99% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 2.
  • yeV is a Toxin of type IV toxin-antitoxin (TA) system.
  • YeV interacts with each of FtsZ and MreB and acts inhibitory. In FtsZ, it inhibits its GTP-dependent polymerization, and in MreB, it inhibits its ATP-dependent polymerization. Since FtsZ and MreB control cell size and morphology, it is known that cells grow when they overexpress yetV, which inhibits them (Molecular microbiology, 2011, 79: 109-118,). And PLoS genetics, 2017, 13: e1007007.).
  • the wild-type yeV has the amino acid sequence shown in SEQ ID NO: 3, and the yeV gene encoding this has, for example, the nucleic acid sequence shown in SEQ ID NO: 4.
  • the nucleic acid sequence encoding the morphogenesis control factor yeV in this embodiment has at least 90%, preferably 93%, 95%, 98% or 99% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 4.
  • the recombinant protein (target protein) in the present embodiment is not particularly limited, and any protein can be used.
  • the target protein means a protein whose purpose is to be recovered and used after being produced using recombinant cells.
  • the target protein include any protein that is preferably produced on an industrial scale, and examples thereof include proteins that can be used for industrial purposes, proteins that can be used for medical purposes, and structural proteins.
  • proteins that can be used for industrial or medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used for vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, etc. Examples include allergens and full-length antibodies or antibody fragments or derivatives.
  • Specific examples of structural proteins include fibroin (for example, spider silk, silk moth silk, etc.), keratin, collagen, elastin, resilin, fragments of these proteins, and proteins derived from them.
  • fibroin includes naturally occurring fibroin and modified fibroin.
  • naturally-derived fibroin means fibroin having the same amino acid sequence as naturally-derived fibroin
  • modified fibroin means fibroin having an amino acid sequence different from that of naturally-derived fibroin. To do.
  • the fibroin may be spider silk fibroin.
  • spider silk fibroin includes natural spider silk fibroin and modified fibroin derived from natural spider silk fibroin. Examples of the natural arachnid fibroin include spider silk proteins produced by arachnids.
  • Fibroin is, for example, a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif. You may.
  • the fibroin according to the present embodiment may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence.
  • the N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.
  • domain sequence refers to a fibroin-specific crystalline region (typically corresponding to (A) n motif of amino acid sequence) and an amorphous region (typically, REP of amino acid sequence).
  • a fibroin-specific crystalline region typically corresponding to (A) n motif of amino acid sequence
  • REP of amino acid sequence
  • the (A) n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
  • the number of amino acid residues of the 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. .. Further, 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, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more. It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues.
  • REP shows an amino acid sequence consisting of 2-200 amino acid residues.
  • REP may 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 plurality of (A) n motifs may have the same amino acid sequence as each other or may have different amino acid sequences.
  • the plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • Naturally derived fibroin for example, a domain sequence represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif can be used.
  • the proteins contained can be mentioned.
  • Specific examples of naturally occurring fibroin include, for example, fibroin produced by insects or arachnids.
  • fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perniya, ⁇ ⁇ (Anteraea pernyi), and tussah.
  • Silk moth (Samia cinthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama), silk moth (Antheraea assama), silk moth (Antheraea assama)
  • Examples include the Hornet silk protein exhaled by the larvae of the silk moth (Vespa similima xanthoptera).
  • insect-produced fibroin include, for example, the silk moth fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders,
  • Spiders belonging to the genus Spider spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne) such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, spiders such as spiders Spiders belonging to (Gasteracantha genus), spiders belonging to the genus Isekigumo (genus Ordgarius) such as Mameitaisekigumo and Mutsutogaysekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc.
  • Trinofundamashi gene Cyrtarachne
  • Torinofundamashi and Otorinofundamashi spiders belonging to the genus Cyrtarachne
  • spiders such as spiders Spiders belonging to (Gasteracantha genus)
  • Spiders belonging to the genus Arachnura spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora ), Spiders belonging to the genus Cyclosa such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders Spiders belonging to the genus Tetragnatha, such as Yasagata spider, Harabiroashidakagumo, and Urokoa shina
  • Examples of the keratin-derived protein include Type I keratin of Capra hircus.
  • a protein containing a domain sequence represented by the formula 3: [REP2] p (where, in formula 3, p represents an integer of 5 to 300.
  • REP2 is Gly-X-.
  • An amino acid sequence composed of Y is shown, and X and Y indicate arbitrary amino acid residues other than Gly.
  • a plurality of REP2s may have the same amino acid sequence or different amino acid sequences.) it can.
  • elastin-derived protein examples include proteins having an amino acid sequence such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine).
  • Examples of the protein derived from resilin include a protein containing a domain sequence represented by the formula 4: [REP3] q (where, in formula 4, q represents an integer of 4 to 300.
  • REP3 is Ser-JJ. It shows an amino acid sequence composed of -Tyr-Gly-U-Pro. J indicates an arbitrary amino acid residue, and it is particularly preferable that it is an amino acid residue selected from the group consisting of Asp, Ser and Thr. U is arbitrary. It is preferable that it is an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser.
  • the plurality of REP4s may have the same amino acid sequence or different amino acid sequences. ) Can be mentioned.
  • the target protein may be a hydrophilic protein or a hydrophobic protein.
  • the sum of the hydrophobicity indexes (hydropathy index, HI) of all the amino acid residues constituting the target protein was obtained, and then the sum was divided by the total number of amino acid residues (mean HI, Hereinafter, it is also referred to as “hydrophobicity”), which is preferably ⁇ 1.0 or higher.
  • hydrophobicity index of amino acid residues a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic karacter of protein7. B. 105-132) is used.
  • the hydrophobicity index of each amino acid is as shown in Table 1 below.
  • the hydrophobicity of the target protein is -0.9 or more, -0.8 or more, -0.7 or more, -0.6 or more, -0.5 or more, -0.4 or more. , -0.3 or more, -0.2 or more, -0.1 or more, 0 or more, 0.1 or more, 0.2 or more, 0.3 or more, or 0.4 or more, and the purpose
  • the hydrophobicity of the 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, but 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 for example, 600 kDa or less, 500 kDa or less, 400 kDa or less. , 300 kDa or less, or 200 kDa or less.
  • the larger the molecular weight of a protein the easier it is to aggregate.
  • the regulatory sequence is a sequence that controls the expression of the recombinant protein (target protein) in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and is appropriately selected according to the type of host. can do.
  • the regulatory sequence may be exogenous or endogenous (host-derived regulatory sequence).
  • the promoter is not limited as long as it functions in the host cell.
  • promoters derived from Escherichia coli or phages such as araBAD promoter, trp promoter (Ptrp), lac promoter, PL promoter, PR promoter and T7 promoter can be mentioned.
  • an artificially designed and modified promoter such as a promoter (Ptrp ⁇ 2) in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, a let I promoter, and a mutant promoter thereof can also be used.
  • the promoter is preferably an inducible promoter, for example, due to the presence of arabinose, an inducible promoter activated by the presence of an inducer (expression inducer) such as isopropyl- ⁇ -thiogalactopyranoside (IPTG).
  • an inducer expression inducer
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • plasmid in which the Shine-Dalgarno sequence, which is a ribosome-binding sequence, and the start codon are adjusted to an appropriate distance (for example, 6 to 18 bases). Transcription termination sequences are not always required, It is preferable to place the transcription termination sequence directly under the gene encoding the target protein.
  • the host As the host (host cell), 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 viewpoint of rapid proliferation and reduction of culture cost, the host cell is preferably a prokaryotic cell such as a bacterium.
  • the host cell may be a coccus, a spiral bacterium, or a bacillus, but is preferably a bacillus.
  • Examples of host cells for prokaryotes such as bacteria include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Vibrio, Pseudomonas and the like. be able to.
  • Preferable examples of prokaryotes include, for example, Escherichia coli, Pseudomonas putida, Pseudomonas fluorescens, Bacillus subtilis, Corynebacterium glutamicum, Brevibacillus, Brevibacillus, and Brevibacillus.
  • the host cell is preferably a microorganism belonging to the genus Escherichia, particularly Escherichia coli.
  • Microorganisms belonging to the genus Escherichia include, for example, Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BLR (DE3) (Merck Millipore), Escherichia coli DH1, Escherichia. ⁇ Kori GI698, Escherichia Kori HB101, Escherichia Kori JM109, Escherichia Kori K5 (ATCC 23506), Escherichia Kori KY3276, Escherichia Kori MC1000, Escherichia Kori MG1655 (ATCC 47076), Escherichia Kori MG1655 (ATCC 47076) 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherich
  • the order of introduction of the target protein expression cassette and the sulA expression cassette does not matter. That is, the target protein expression cassette may be incorporated into the host cell into which the sulA expression cassette has been introduced, or the sulA expression cassette may be incorporated into the host cell into which the target protein expression cassette has been introduced.
  • Recombinant cells containing the target protein expression cassette can be obtained, for example, by transforming the host cell with an expression vector containing at least the nucleic acid sequence encoding the target protein.
  • the expression vector may contain a target protein expression cassette.
  • the recombinant cell according to the present embodiment may have a target protein expression cassette extrachromosomally, or may have a target protein expression cassette on the chromosome (on the genome), but the target protein expression It is preferable that the cassette is on a chromosome (on the genome).
  • Recombinant cells containing the sulA expression cassette can be produced in the same manner.
  • a known method can be used, and examples thereof include transforming a host cell with a plasmid vector.
  • a known method can be used as a method for incorporating the target protein expression cassette into the genome.
  • a method of incorporating the target protein expression cassette into the host chromosome using the mechanism of lysogenization of HK022 phage, homologous recombination of ⁇ 80 phage.
  • the ⁇ red method applying the recombination mechanism in double-strand break repair of ⁇ phage, the Red / ET homologous recombination method, and the transposon activity using pUT-mini Tn5.
  • the transfer method that has been performed can be mentioned.
  • the target protein expression cassette can be incorporated into the genome of the host cell according to the method described in the kit by using "Transposon Gene Transfer Kit: pUTmini-Tn5 Kit” manufactured by Biomedal.
  • the target protein expression cassette is operably linked to one or more regulatory sequences on the genome of the host cell by recombining the DNA fragment containing at least the nucleic acid sequence encoding the target protein into the genome of the host cell. It may be incorporated above.
  • a target protein expression cassette is hosted by an integrase of a ⁇ phage via an attachment site (attB site) on the genomic DNA of the host cell and an attachment site (attP site) on a vector.
  • an attachment site attB site
  • an attachment site attP site
  • a method of incorporating the target protein expression cassette into the genomic DNA of the host cell is preferable.
  • any method for introducing DNA into the host cell can be used.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • the protoplast method Japanese Patent Laid-Open No. 63-248394
  • the method described in Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979), etc. Can be mentioned.
  • Transformation of microorganisms belonging to the genus Brevibacillus is carried out, for example, by the method of Takahashi et al. (J. Bacteriol., 1983, 156: 1130-1134) or the method of Takagi et al. (Agric. Biol. Chem., 1989, 53:30). -3100), or by the method of Okamoto et al. (Bioscii. Biotechnol. Biochem., 1997, 61: 202-203).
  • the type of vector used for transformation can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. ..
  • the vector include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 ().
  • QIAGEN pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol.
  • pGKA2 prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Patent Application Laid-Open No. 60-221091 Gazette, pTerm2 (US Pat. No. 4,686,191, US Pat. No. 4939094, US Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. , 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), and the like.
  • pUC18 When Escherichia coli is used as the host cell, pUC18, pBluescriptII, pSupex, pET22b, pCold and the like can be mentioned as suitable vectors.
  • 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. 4-278901), pHY4831 (J). .Vector., 1987, 1239-1245), pNU200 (Shigezo Utaka, Journal of Japan Society for Bioscience and Biotechnology, 1987, 61: 669-676), pNU100 (Appl. Microbial. Biotechnol., 1989, 30: 75-80), pNU211 (J.
  • pNU211R2L5 Japanese Patent Laid-Open No. 7-170984
  • pNH301 Appl. Environ. Microbiol., 1992, 58: 525-531
  • pNH326, pNH400 J. Biochem. Vector., 1995, 177: 745-749
  • pHT210 Japanese Patent Laid-Open No. 6-133782
  • pHT110R2L5 Appl. Microbiol. Biotechnol., 1994, 42: 358-363
  • PNCO2 Japanese Patent Laid-Open No. 2002-238569
  • the method for producing a recombinant protein according to the present embodiment includes at least a production process.
  • the production step is a step of culturing the above-mentioned recombinant cells in a protein production medium.
  • the method for producing a recombinant protein according to the present embodiment may further include a pre-culture step of culturing the above-mentioned recombinant cells in a pre-culture medium before the production step.
  • the protein production medium for culturing the recombinant cells is not particularly limited, and can be selected from known natural media or synthetic media depending on the type of the recombinant cells.
  • As the protein production medium for example, carbon source, nitrogen source, phosphoric acid source, sulfur source, vitamins, minerals, nutrients required by auxotrophy, and other components selected from various organic and inorganic components are used. If necessary, a liquid medium contained therein can be used. Those skilled in the art may appropriately set the type and concentration of the medium component.
  • the protein production medium preferably contains naturally occurring components.
  • the naturally derived component means a component such as a natural product (for example, yeast) itself or an extract from the natural product (for example, Yeast Extract).
  • Naturally-derived ingredients are usually those in which the types of ingredients contained and the content of each are not completely specified.
  • Naturally-derived components include, for example, at least one selected from the group consisting of vitamins, low molecular weight peptides (eg, peptides having 2 to 20 amino acid residues) and amino acids.
  • Examples of the carbon source include sugars such as glucose, sucrose, lactose, galactose, fructose and a hydrolyzate of starch, alcohols such as glycerol and sorbitol, and organic acids such as fumaric acid, citric acid and succinic acid.
  • sugars such as glucose, sucrose, lactose, galactose, fructose and a hydrolyzate of starch
  • alcohols such as glycerol and sorbitol
  • organic acids such as fumaric acid, citric acid and succinic acid.
  • the carbon source may be one type or two or more types of carbon sources may be mixed at an arbitrary ratio.
  • the concentration of the carbon source in the protein production medium is about 0.1 w / v% to 50 w / v%, preferably about 0.5 w / v% to 40 w / v%, and more preferably 1 w / v% to 30 w / v%.
  • the degree particularly preferably about 5 w / v% to 20 w / v%.
  • glycerol or glucose is preferably used as the carbon source, and glycerol or glucose may be mixed with another carbon source in an arbitrary ratio.
  • the ratio of glycerol or glucose in the carbon source is preferably 10% by weight or more, more preferably 50% by weight or more, and particularly preferably 70% by weight or more.
  • the preferable initial concentration of the carbon source at the start of the culture is as described above, but the carbon source may be appropriately added depending on the consumption of the carbon source during the culture.
  • the nitrogen source examples include inorganic nitrogen salts such as nitrates, ammonium salts, ammonia gas and aqueous ammonia, amino acids, peptones, extracts, and organic nitrogen sources such as corn steep liquor (CSL), which is a by-product of the cornstarch manufacturing industry.
  • examples of peptones include casein peptone, meat peptone, myocardial peptone, gelatin peptone, soybean peptone and the like.
  • the extracts include meat extract, yeast extract, heart exudate (heart infusion) and the like.
  • the amino acid or the nitrogen source containing the peptide it is preferable that the content of the lower molecular weight peptide and the amino acid is high.
  • Examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphoric acid polymers such as pyrophosphoric acid.
  • sulfur source examples include inorganic sulfur compounds such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione.
  • vitamins examples include biotin, choline chloride, cyanocobalamin, folic acid, inositol, nicotinic acid, 4-aminobenzoic acid, pantothenic acid, pyridoxine, riboflavin, thianmin, chimdin and the like.
  • sources of vitamins include various extracts such as malt extract, potato extract and tomato juice.
  • Examples of minerals include sulfur (S), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), sodium (Na), and the like, in addition to phosphorus (P).
  • the culture in the production step can be aerobically carried out by, for example, aeration culture or shaking culture.
  • the culture can be carried out by batch culture (batch culture), fed-batch culture (fed-batch culture), continuous culture (continuous culture), or a combination thereof.
  • the pH of the protein production medium may be, for example, 3.0 to 9.0.
  • the culture temperature may be, for example, 15-40 ° C.
  • the culturing time may be, for example, 1 to 60 hours.
  • the culture conditions are not particularly limited as long as the recombinant cells can proliferate and the target protein can be accumulated in the recombinant cells expressing the target protein.
  • the recombinant cells may or may not proliferate during the period in which the target protein is expressed.
  • the culture conditions may or may not be the same in the period before the expression of the target protein and the period after the start of expression.
  • Culture temperature usually has a large effect on cell proliferation.
  • the lower limit temperature of growth is 0 ° C. or slightly lower than the freezing temperature of water in cells
  • the upper limit temperature is determined by the denaturation temperature of polymer compounds such as proteins and nucleic acids.
  • the temperature range in which a certain strain can grow is relatively narrow. For example, in Escherichia coli, the lower limit temperature for growth is 0 to 15 ° C, the upper limit is 46 ° C, and the optimum temperature for growth is around 36 to 42 ° C.
  • thermophilic bacteria with an optimum temperature of 20 ° C or lower
  • thermophilic bacteria with an optimum temperature of 20 to 45 ° C
  • thermophiles with an optimum temperature of 45 ° C or higher.
  • the optimum growth temperature means the temperature at which the microorganism to be cultured can obtain the maximum specific growth rate
  • the specific growth rate means the growth rate per unit amount of microorganisms, which is a value peculiar to the microorganisms. It changes depending on the culture conditions.
  • the "optimal growth temperature” means that the microorganism obtains 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. The temperature at which it can be produced.
  • the recombinant cell when the recombinant cell is expressing the target protein (after induction of expression when the expression of the target protein is inducible), the recombinant cell is proliferated by adjusting the culture temperature or the like. By cooling or maintaining the recombinant cells at a temperature lower than the optimum temperature, the expression level of the target protein in the recombinant cells can be increased.
  • the temperature lower than the optimum growth temperature of the recombinant cells may be, for example, a temperature 3 to 25 ° C. lower than the lower limit of the optimum growth temperature of the recombinant cells, and 8 to 20 ° C. lower. Well, it may be 10-18 ° C lower temperature, 12 ° C-18 ° C lower temperature, 14 ° C-17 ° C lower temperature, 3-10 ° C lower temperature, 5-10 ° C lower temperature. The temperature may be as low as 8 ° C.
  • the recombinant cell according to the present embodiment may be one capable of inducing the expression of the target protein and sulA. Induction of expression of the target protein and sulA is carried out by activating transcription by an inducible promoter (transcription of the nucleic acid encoding the target protein). Activation of the inducible promoter can be carried out according to a method known in the art, depending on the type of inducible promoter.
  • the induction expression timing of sulA may be 6 hours before and after the induction expression of the target protein, 4 hours before and after, or 2 hours before and after. It is preferable to induce the expression of sulA at the same time as or after the induction of the expression of the target protein.
  • an inducible promoter activated by the presence of an inducer such as isopropyl- ⁇ -thiogalactopyranoside (IPTG) or arabinose
  • the inducer should be added to the culture medium.
  • the inducer may be added to the culture broth at one time or in multiple batches, or may be added to the culture broth by continuous feed.
  • the fed-batch substrate solution may contain an inducer and be fed.
  • the amount of the inducible substance to be added can be set according to the type of the inducible substance and the inducible promoter, and for example, in IPTG, it can be in the range of 0.01 to 10 mM per culture medium, and is preferable. , 0.05 to 5 mM, more preferably 0.05 to 2 mM.
  • the range can be in the range of 0.01 to 1 M per culture medium, preferably in the range of 0.05 to 0.5 M.
  • the expression of the recombinant protein can be induced by increasing or decreasing the temperature of the culture solution.
  • the expression of recombinant protein during proliferation is suppressed by setting the temperature of the culture medium during proliferation in the range of 20 to 37 ° C. Then, the expression of the recombinant protein can be induced by raising the temperature of the culture medium to 38 to 44 ° C.
  • the pH of the culture solution at the time of growth was set to 6.5 to 7.5 as described in JP-A-6-292563, and the expression of the recombinant protein was induced.
  • the pH of the culture solution was set to 4.5 to 6.5 at the start, more stable expression induction can be performed.
  • the time of transition from the stage of proliferating the recombinant cells to the stage of inducing the expression of the recombinant protein is not particularly limited and can be appropriately set according to the configuration of the culture system and the design of the production process. From the viewpoint of efficient production of the recombinant protein, it is preferable to start the induction of the expression of the recombinant protein when the proliferation of the recombinant cells reaches the metaphase to the late stage of the logarithmic growth phase.
  • Proliferation of recombinant cells begins in the delayed or inducing phase (the period when the number of cells increases slowly in the early stage of culture), and goes through the logarithmic growth phase (the period when the number of cells doubles and logarithmically increases per unit time). , Reach the stationary phase (the period when the net number of cells does not change).
  • the middle stage of the logarithmic growth phase refers to the period when the number of cells reaches an intermediate level between the number of cells in the delayed phase and the number of cells in the stationary phase
  • the latter stage of the logarithmic growth phase refers to the period from the middle stage to the stationary phase.
  • the time when the induction of the expression of the recombinant protein is started for example, in the case of a recombinant cell in which the value of OD600 in the stationary phase reaches about 150, the time when the value of OD600 reaches 30 to 110 is the time. It is preferable that the time reaches 40 to 90, more preferably 50 to 80, and more preferably 50 to 80.
  • the time for inducing the expression of the recombinant protein may be set until the set production amount is reached, depending on the host used and the type of the target protein. Since the production rate changes depending on the culture conditions such as the temperature of the culture solution, it is not necessary to uniquely determine the time for inducing the expression of the recombinant protein.
  • the time for inducing the expression of the recombinant protein may be set according to the progress of the separation and purification of the recombinant protein in the next step. Further, in industrial production, it is preferable to set a time for inducing the expression of the recombinant protein so as not to affect the proliferation of the recombinant cells and the transfer of the grown recombinant cells in parallel. ..
  • the pre-culture step is a step of culturing recombinant cells in a pre-culture medium before the production step.
  • the specific embodiment of the pre-culture medium is the same as that described in the above-mentioned protein production medium.
  • the method for producing a recombinant protein it is preferable to use a medium rich in nutritional components as a pre-culture medium rather than a protein production medium. As a result, the production amount of the target protein per cell can be further increased.
  • the expression construct of the present embodiment is different from the first expression cassette containing a nucleic acid sequence encoding a recombinant protein operably linked to the first promoter, and the first promoter. It has a second expression cassette containing a nucleic acid sequence encoding sulA operably linked to a second promoter.
  • the first promoter and the second promoter can be the above promoters
  • the first expression cassette can be the target protein expression cassette
  • the second expression cassette can be the sulA expression cassette.
  • Example 1 (1) Preparation of Escherichia coli strain inducing and expressing modified fibroin (target protein) Nucleotide sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes and Based on the amino acid sequence, a modified fibroin (hereinafter, also referred to as “PRT966”) having the amino acid sequence shown by SEQ ID NO: 5 was designed.
  • the amino acid sequence shown in SEQ ID NO: 5 has an amino acid sequence in which amino acid residues are substituted, inserted and deleted for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes, and further.
  • the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 6 is added to the N-terminal.
  • nucleic acid encoding PRT966 was synthesized. An NdeI site was added to the nucleic acid at the 5'end, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombined into a pET-22b (+) vector to obtain a pET-22 (+) / PRT966 vector.
  • the first modified fibroin expression cassette was integrated onto the host chromosome using the mechanism by which HK022 phages lysogenize.
  • the mechanism is a sequence-specific recombination between a specific site on the host chromosome (atB site) and a specific site on the phage genome (atP (HK022) site).
  • FIG. 1 is a schematic diagram showing an outline of a method for incorporating a modified fibroin expression cassette onto a host chromosome by utilizing the mechanism by which HK022 phages are lysogenized.
  • a nucleic acid encoding PRT966 was excised from the pET-22 (+) / PRT966 vector by restriction enzyme treatment with NdeI and EcoRI, and then recombined into a plasmid vector attHK022-Cm2 having an attP (HK022) site to attHK022-.
  • a T7p-PRT966-T7t-FRT-Cm2-ori_R6K-FRT vector was obtained.
  • the attHK022-T7p-PRT966-T7t-FRT-Cm2-ori_R6K-FRT vector was introduced into the host and sequence-specific between the attB site on the host chromosome and the attP (HK022) site of the same vector.
  • a modified fibroin (PRT966) expression cassette was recombinantly integrated onto the host chromosome.
  • a helper plasmid pAH69 J. Bact 183: 6384-6393 having an int gene was introduced into the host in advance to express the integrase.
  • the helper plasmid pCP20 Proc. Natl. Acad. Sci. USA, 97: 6640-6645
  • the second modified fibroin expression cassette was integrated onto the host chromosome using the mechanism by which ⁇ 80 phage is lysogenized.
  • the mechanism is a sequence-specific recombination between a specific site of the host chromosome (atB site) and a specific site of the phage genome (atP ( ⁇ 80) site).
  • FIG. 2 is a schematic diagram showing an outline of a method for incorporating a modified fibroin expression cassette onto a host chromosome by utilizing the mechanism of lysogenization of ⁇ 80 phage.
  • a nucleic acid encoding PRT966 was excised from the pET-22 (+) / PRT966 vector by restriction enzyme treatment with NdeI and EcoRI, and then recombined into a plasmid vector att ⁇ 80-Km1_1 having an attP ( ⁇ 80) site to att ⁇ 80-.
  • An ori_R6K-FRT-Km1-FRT-SPT3p-PRT966-T7t-FRT vector was obtained.
  • the att ⁇ 80-ori_R6K-FRT-Km1-FRT-SPT3p-PRT966-T7t-FRT vector was introduced into the host into which the first modified fibroin expression cassette was incorporated by the method (a) above, and the vector was introduced onto the host chromosome.
  • a second modified fibroin (PRT966) expression cassette was integrated onto the host chromosome by sequence-specific recombination between the attB site and the attP ( ⁇ 80) site of the same vector.
  • the helper plasmid pCP20 was introduced to express FLP, thereby removing the kanamycin resistance gene sandwiched between the FRT sequences.
  • (C) ⁇ Red_manX The third modified fibroin expression cassette was integrated on the host chromosome using the homologous recombination system possessed by ⁇ phage.
  • the homologous recombination system causes homologous recombination by the exo, bet, and gam gene products in the Red region of the phage genome.
  • FIG. 3 is a schematic diagram showing an outline of a method for incorporating a modified fibroin expression cassette onto a host chromosome by utilizing the homologous recombination system possessed by ⁇ phage.
  • a modified fibroin expression cassette (manX5'homologous sequence-SPT3 promoter-PRT966-T7 terminator) is contained in this order by a PCR method using a primer that introduces a modification into the T7 promoter using the pET-22 (+) / PRT966 vector as a template. ) was amplified.
  • the chloramphenicol resistance gene expression cassette (including the T7 terminator homologous sequence-FRT-chloramphenicol resistance gene-FRT-manX3'homologous sequence in this order) is amplified by the PCR method using the pKD13-Cm vector as a template. did. Both PCR products were ligated using an In-Fusion® cloning system (manufactured by Takara Bio Inc.). Next, a DNA fragment ligated to the host incorporating the first modified fibroin expression cassette and the second modified fibroin expression cassette by the methods (a) and (b) above is introduced, and manX5 on the host chromosome is introduced.
  • a fibroin (PRT966) expression cassette was integrated on the host chromosome.
  • a helper plasmid pKD46 Proc. Natl. Acad. Sci. USA, 97: 6640-6645
  • the helper plasmid pCP20 was introduced to express FLP to remove the chloramphenicol resistance gene sandwiched between the FRT sequences.
  • sulA_F 5'-TTTAAGAAGGAGATATACATATGTACACTTCAGGCTATGCAC-3'(SEQ ID NO: 7)
  • sulA_R 5'-TGTCGACGGAGCTCGAATTCTTAATGATACAAATTAGAGTGAATTTTTAGCCCGG-3' (SEQ ID NO: 8)
  • RBS-4 5'-ATGTATATCTCCTTCTTAAGT -GAATTCGAGCTCCGTCGAC-3'(SEQ ID NO: 10)
  • PCR for amplifying sulA is performed by adding a primer having a final concentration of 0.2 ⁇ M and a BL21 (DE3) cell suspension having an OD to 0.01 according to the attached manual, and using PrimeSTAR® Max (manufactured by Takara Bio Co., Ltd.). ), And the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 30 seconds were performed for 30 cycles.
  • a 1 ng attP21-KmR2 vector was obtained using a primer having a final concentration of 0.2 ⁇ M and PrimeSTAR® Max (manufactured by Takara Bio Inc.).
  • As a template 30 cycles of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 30 seconds were performed.
  • the nucleotide sequence of the obtained expression vector was confirmed by the Sanger method.
  • AttP21-T7p- using recombinant cells having three modified fibroin expression cassettes obtained by the method (1) on the host chromosome as host cells.
  • the sulA-T7t-FRT-KmR2-ori_R6K-FRT vector is introduced into the host, and the sulA expression cassette is hosted by sequence-specific recombination between the attB site on the host chromosome and the attP (P21) site of the same vector.
  • a helper plasmid pAH121 J. Bact 183: 6384-6393
  • modified fibroin expression of modified fibroin protein
  • Recombinant cells obtained by the methods (1) and (2) above having three modified fibroin expression cassettes on the chromosome and expressing sulA
  • sulA-induced expression strain were cultured by the following method, and the expression level of modified fibroin was analyzed.
  • the recombinant cell obtained by the method (1) above recombinant cell having three modified fibroin expression cassettes on three chromosomes and not having a sulA expression cassette.
  • Control strain also referred to as "Control strain”
  • the Control strain and the sulA-induced expression strain were each cultured in 2 mL of LB medium for 15 hours.
  • the culture solution was added to 100 mL of the preculture medium (seed culture medium in Table 2) so that the OD600 was 0.005.
  • the temperature of the culture solution was kept at 30 ° C., and flask culture was carried out until the OD600 reached 5 (about 15 hours) to obtain a seed culture solution.
  • the seed culture medium was added to a jar fermenter to which 500 mL of protein production medium (production medium in Table 3) was added so that the OD600 was 0.05.
  • the temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 30 to 40% of the dissolved oxygen saturation concentration.
  • the feed solution (the feed substrate solution in Table 4) was added at a rate of 6 g / hour.
  • the temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 16 hours.
  • 1 M of isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 0.1 mM to induce expression of modified fibroin and sulA.
  • SDS-PAGE was performed using cells prepared from the culture solutions before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of the desired modified fibroin-sized band depending on the addition of IPTG. did.
  • the mixture was stirred with a shaker (manufactured by Tietech Co., Ltd., 200 rpm, 37 ° C.) for 60 minutes. Then, it was centrifuged (11,000 g, 30 minutes, room temperature) with the above-mentioned Kubota centrifuge, and the supernatant was discarded to obtain SDS-washed granules (precipitate). 4) The SDS washed granules were suspended in a DMSO solution containing 1 M lithium chloride so as to have a concentration of 100 mg / mL, and heat-treated at 80 ° C. for 1 hour.
  • the obtained freeze-dried powder was subjected to polyacrylamide gel electrophoresis, and a band of the target protein was detected by an appropriate method.
  • Image analysis was performed using Totallab (nonliner dynamics ltd.), And the production amount of modified fibroin was evaluated.
  • the culture solution containing the collected cells was diluted to an appropriate concentration, and the average diameter and particle concentration of the particles were measured using a particle counting analyzer CDA-1000 (Cysmex Co., Ltd.), and each of them was measured. The average diameter of cells and the cell concentration were used.
  • the production amount of modified fibroin per cell was calculated from the production amount of modified fibroin by image analysis and the number of cells calculated from the cell concentration, and the relative value when the value of the induction time of 28 hours in the Control strain was set to 100% was calculated. ..
  • FIG. 4 is a graph showing the relative value of the average cell diameter of the sulA-induced expression strain based on the Control strain. As shown in FIG. 4, the average cell diameter of the sulA-induced expression strain increased to 115% from the start of the induction expression as compared with the Control strain.
  • FIG. 5 is a graph showing a relative value of the modified fibroin production amount (production amount per cell) of the sulA-induced expression strain based on the Control strain. As shown in FIG. 5, the amount of modified fibroin produced by the sulA-induced expression strain increased to about 200% 24 and 28 hours after the expression induction as compared with the Control strain.
  • the protein-encoding region (CDS) of the yeV gene was amplified by PCR using the following primers, yeV_F and yeV_R.
  • the plasmid vector attP21-KmR2 having an attP (P21) site was linearized and amplified by PCR using the following primer sets of RBS-4 and pET-MCS_F. These two amplified fragments were concatenated using NEBiller HiFi DNA Assembury Master Mix (New England Biolab Japan Co., Ltd.) according to the attached manual, and attP21-T7p-yeV-T7t-FRT-KmR2-ori_R6K-FRT vector.
  • a primer with a final concentration of 0.2 ⁇ M and a BL21 (DE3) cell suspension having an OD to 0.01 are attached according to the attached manual, and PrimeSTAR® Max (manufactured by Takara Bio Co., Ltd.) ), And the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 30 seconds were performed for 30 cycles.
  • a 1 ng attP21-KmR2 vector was obtained using a primer having a final concentration of 0.2 ⁇ M and PrimeSTAR® Max (manufactured by Takara Bio Inc.).
  • yeeV_F 5'-TTTAAGAAGGAGATATACATatgaacacattacccgacacac-3'(SEQ ID NO: 11)
  • yeeV_R 5'-TGTCGACGGAGCTCGAATTCtcatcgtttctccggatgct-3' (SEQ ID NO: 12)
  • RBS-4 5'-ATGTATATCTCCTTCTTAAAGTTAAACA3' -GAATTCGAGCTCCGTCGAC-3'(SEQ ID NO: 14)
  • the YeV expression cassette was integrated onto the host chromosome in the same manner as in the method for integrating the expression cassette of the morphogenesis control factor of Example 1 onto the host chromosome.
  • modified fibroin Recombinant cells obtained by the methods (1) and (2) above recombinant cells having three modified fibroin expression cassettes on three chromosomes and having a yesV expression cassette.
  • it is also referred to as “yeV-induced expression strain”
  • the expression level of modified fibroin was analyzed by culturing in the same manner as in Example 1.
  • the recombinant cell obtained by the method (1) above recombinant cell having three modified fibroin expression cassettes on three chromosomes and not having a YeV expression cassette; hereinafter, also referred to as "Control strain”
  • Control strain was evaluated in the same way.
  • FIG. 6 is a graph showing the relative value of the average cell diameter of the YeV-induced expression strain based on the Control strain. As shown in FIG. 6, the average cell diameter of the YeV-induced expression strain increased to 113% from the start of the induction expression as compared with the Control strain.
  • FIG. 7 is a graph showing a relative value of the modified fibroin production amount (production amount per medium) of the YeV-induced expression strain based on the Control strain. As shown in FIG. 7, the amount of modified fibroin produced by the YeV-induced strain was reduced to 94% 24 hours after the induction of expression and 91% 28 hours after the induction of expression, as compared with the Control strain.
  • Example 1 Conclusion From the results of Example 1 and Test Examples, the production amount does not increase if the cells are enlarged, but the production amount of the target protein is to increase the cells by taking the induced expression of sulA as a means. It is considered to be important for the increase of.
  • Example 2 (1) Preparation of modified fibroin-producing strain carrying an arabinose-inducible sulA expression plasmid GFP CDS mounted on pGLO (registered trademark, Bio-Rad Laboratories, Inc.) was replaced with sulA CDS, and pGLO was used. -SulA was prepared. sulA was amplified by PCR using 1 ng of the attP21-T7p-sulA-T7t-FRT-KmR2-ori_R6K-FRT vector constructed in Example 1 as a template and the following primer sets of sulA_F and ara-sulA_R.
  • sulA_F 5'-TTTAAGAAGGAGATATACATATGTACACTTCAGGCTATGCAC-3'(SEQ ID NO: 7)
  • ara-sulA_R 5'-ACCGAGCTCGAATTCttaatgatacaaattagagt-3' (SEQ ID NO: 15)
  • RBS-4 5'-ATGTATATCTCCTTCTTAAAGTTAAACAAA-3'(SEQ ID NO: 9) 5'-GAATTCGAGCTCGGTACCCG-3'(SEQ ID NO: 16)
  • PCR was carried out using PrimeSTAR (registered trademark) Max (manufactured by Takara Bio Inc.) under the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 30 seconds for 30 cycles.
  • the two obtained fragments were concatenated using NEBiller HiFi DNA Assembury Master Mix (New England Biolab Japan Co., Ltd.) according to the attached manual to prepare pGLO-sulA.
  • the nucleotide sequence of the obtained expression vector was confirmed by the Sanger method.
  • the prepared pGLO-sulA is transformed into an Escherichia coli strain that induces and expresses the modified fibroin prepared in Example 1, and has recombinant cells (having three modified fibroin expression cassettes on chromosomes and having a sulA expression cassette). Recombinant cells (hereinafter, also referred to as “sulA-induced expression strain”) were obtained. Recombinant cells having three modified fibroin expression cassettes on the chromosome and no sulA expression cassette were designated as "Control strains”.
  • modified fibroin (2) Expression and evaluation of modified fibroin
  • the expression of modified fibroin was quantified by the same culture method and evaluation method as in Example 1, and the cell size was measured. However, 2% (0.13M) of L-arabinose was used instead of IPTG to induce the expression of sulA.
  • the timing of inducing and expressing sulA is three time points: the timing of inducing modified fibroin with IPTG (T0) 4 hours earlier (-4h), the timing of simultaneous T0 (0h), and the timing of 2 hours later than T0 (+ 2h). I went there. Control did not induce arabinose.
  • Results Table 5 shows the relative values of the average cell diameter with respect to Control at each induction time. Regardless of the timing of -4h, 0h, or + 2h, the cells were larger in T16 to T28 in which the modified fibroin was induced and expressed, as compared with Control.
  • Table 6 shows the relative values of the production amount per medium based on the Control of each induction time. An increase of 10% or more was observed in the production volume at T20 at 0h and + 2h.

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Abstract

La cellule recombinée selon la présente invention contient une séquence d'acide nucléique codant pour une protéine recombinée et une ou plusieurs séquences régulatrices liées de manière fonctionnelle à la séquence d'acide nucléique et, de plus, une séquence d'acide nucléique codant pour la sulA lui ayant été introduite. Le procédé selon la présente invention pour produire une protéine recombinée comprend une étape de production pour la culture de cellules recombinées dans un milieu de production de protéine, lesdites cellules recombinées contenant une séquence d'acide nucléique codant pour la protéine recombinée et une ou plusieurs séquences régulatrices liées de manière fonctionnelle à la séquence d'acide nucléique et, de plus, ayant une séquence d'acide nucléique codant pour un régulateur de la morphogenèse sulA lui ayant été introduit.
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