JP2020080727A - Methods for producing proteins of interest - Google Patents

Abstract

To provide methods for producing proteins of interest including accumulation of the proteins of interest in an insoluble fraction in the cell.SOLUTION: Disclosed is a method for producing a protein of interest comprising culturing a recombinant cell expressing the protein of interest in the presence of a lower alcohol to cause accumulation of the protein of interest in an insoluble fraction in the cell.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a target protein.

The gene recombination technology has enabled large-scale production of useful proteins derived from various species including human beings using microorganisms such as Escherichia coli as a host.

It is known that proteins heterologously expressed in Escherichia coli or the like form insoluble aggregates (inclusion bodies) in the cytoplasm because they are not correctly folded (Non-patent Document 1). In particular, it is considered that proteins rich in hydrophobic amino acids form inclusion bodies in the cytoplasm due to heterologous expression, and their expression in the soluble fraction tends to be extremely low or not to occur at all. Therefore, there is a problem that it is difficult to produce a protein using the soluble fraction.

Groot et al. Futuremicrobiology(2008),3(4),423-435

However, protein production using microorganisms such as Escherichia coli is considered to be the cheapest and simplest production system, and since the formed inclusion bodies are protected from degradation by protease, the inclusion bodies are If the target protein of high purity is contained, there is an advantage that purification itself is easy.

Then, this invention aims at providing the manufacturing method of the target protein including accumulating the target protein in an insoluble fraction.

The present invention relates to the following inventions, for example.
[1]
A method for producing a target protein, comprising:
A method comprising culturing a recombinant cell expressing a target protein in the presence of a lower alcohol to accumulate the target protein in an insoluble fraction.
[2]
The method according to [1], wherein the expression of the target protein is inducible.
[3]
The method according to [1] or [2], wherein 80% or more of the total expression amount of the target protein expressed by the recombinant cell is accumulated in the insoluble fraction.
[4]
The method according to any of [1] to [3], wherein the target protein has a hydrophobicity of -1.0 or more.
[5]
The method according to any one of [1] to [4], which comprises cooling or maintaining the recombinant cells at a temperature lower than the optimum growth temperature of the recombinant cells.
[6]
The method according to any one of [1] to [5], wherein the lower alcohol is ethanol.
[7]
The method according to any one of [1] to [6], wherein the target protein is a structural protein.
[8]
The method according to any one of [1] to [7], wherein the target protein is fibroin.
[9]
The method according to any one of [1] to [8], wherein the target protein is spider silk fibroin.
[10]
The method according to any one of [1] to [9], wherein the recombinant cell is a bacterial cell of the genus Escherichia.
[11]
A method of accumulating a target protein in an insoluble fraction by culturing expressing recombinant cells having a gene encoding the target protein in the presence of lower alcohol.

According to the present invention, it is possible to provide a method for producing a target protein, which comprises accumulating the target protein in the insoluble fraction. Increasing the amount of target protein in the insoluble fraction by culturing recombinant cells expressing the target protein in the presence of inexpensive lower alcohol without modifying the host or changing culture conditions significantly Therefore, the productivity of the target protein can be increased.

It is a stained photograph which shows the result of having confirmed the expression of the target protein by polyacrylamide gel electrophoresis (SDS-PAGE). For staining, His-tag staining using an Invision staining solution was performed. The first to third columns from the left show the migration results of the sample derived from the cells cultured at 20° C. after the addition of IPTG, and the fourth column shows the migration results of the protein molecular weight marker. -1% and 3% indicate the final concentration of EtOH added (- indicates not added). The arrow indicates the position of the band based on the size of the target protein. 2 is a staining photograph showing the result of confirming the expression of the target protein in each solution of the soluble fraction and the insoluble fraction by SDS-PAGE as in FIG. 1. Sup (rows 1 to 3) shows a soluble fraction, and Ppt (rows 4 to 6) shows an insoluble fraction. -, 1% and 3% indicate the final concentration of EtOH added (- indicates not added). The seventh column shows the results of migration of protein molecular weight markers. The arrow indicates the position of the band based on the size of the target protein.

Hereinafter, modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Method for producing target protein]
The method for producing a target protein according to one embodiment of the present invention includes culturing a recombinant cell expressing the target protein in the presence of a lower alcohol to accumulate the target protein in the insoluble fraction.

(Recombinant cell)
The recombinant cell according to one embodiment of the present invention expresses a target protein. A recombinant cell means a cell that has been recombinantly engineered to express a protein of interest.

The recombinant cell according to one embodiment of the present invention can be obtained, for example, by introducing an expression cassette for expressing a target protein. Examples of the expression cassette include an expression cassette having a nucleic acid sequence encoding a protein of interest and one or more regulatory sequences operably linked to the nucleic acid sequence.

Examples of the method of introducing the expression cassette into the host cell include a method of transforming the expression vector into which the expression cassette has been introduced into the host cell, and a method of directly incorporating the expression vector or the present expression cassette into the genomic DNA of the host cell. Be done. The expression cassette for the protein of interest is preferably integrated into the genomic DNA of the host cell.

As a method of transforming an expression vector into which an expression cassette has been introduced into a host cell, a known method can be used, and examples include transforming the expression vector into a host cell using a plasmid. For example, recombinant Escherichia coli can be produced by introducing the expression cassette into Escherichia coli by the above method.

As a method for incorporating the above-mentioned expression vector or expression cassette into the genomic DNA of a host cell, known methods can be used. For example, the λred method applying the recombination mechanism in the double-strand break repair of λ phage, Red/ The ET homologous recombination method and the transposition method utilizing the transposon activity using pUT-mini Tn5 can be mentioned. In addition, for example, a gene transfection kit using transposon: pUTmini-Tn5 Kit of Biomedal Co., Ltd. can be used to incorporate the expression cassette into the genomic DNA of the host cell according to the method described in the kit.

As a 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, the host cells are preferably prokaryotic cells such as bacteria from the viewpoints of rapid growth and reduction of culture costs.

Examples of prokaryotic host cells such as bacteria include microorganisms belonging to Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. .. Preferred examples of prokaryotes include E. coli, Bacillus subtilis, Pseudomonas, Corynebacterium, and Lactococcus. The host cell is preferably a bacterium of the genus Escherichia, in particular 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 No. 47076, Escherichia coli. 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia W3185 (Escherichia W3) ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue and the like. The host cell is preferably Escherichia coli.

As a method for introducing the present expression cassette into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP-A-63-248394), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979). Can be mentioned.

Transformation of a microorganism belonging to the genus Brevibacillus is performed, 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, 53:3099). -3100), or the method of Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61:202-203).

The type of vector (hereinafter simply referred to as “vector”) into which the present expression cassette is introduced may be appropriately selected according to the type of host, such as plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, etc. it can. Examples of the vector include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (PQE-8). Manufactured by QIAGEN), pKYP10 (JP-A-58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [EscherrJsPriSpricheria]. )), pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), JP-A-60-221091. Gazette], pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. 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.

When E. coli is used as the host cell, pUC18, pBluescriptII, pSupex, pET22b, pCold and the like can be mentioned as suitable vectors.

Specific examples of a vector suitable for a microorganism belonging to the genus Brevibacillus include pUB110, which is known as a Bacillus subtilis vector, or pHY500 (JP-A-2-31682), pNY700 (JP-A-4-278091), pHY4831 (J Bacteriol., 1987, 1239-1245), pNU200 (Shigezo Utaka, Journal of Japan Society of Agricultural Chemistry 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), pNU211. (J. Biochem., 1992, 112:488-491), pNU211R2L5 (JP-A-7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58:525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177: 745-749), pHT210 (JP-A-6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363), or microorganisms belonging to Escherichia coli and Brevibacillus. And pNCO2 (Japanese Patent Laid-Open No. 2002-238569), which is a shuttle vector of

Examples of eukaryotic host cells include yeast and filamentous fungi (mold and the like).

As yeast, for example, Saccharomyces genus, Schizosaccharomyces genus, Kluyveromyces genus, Trichosporon (Trichospida) genus, Schwanniomyces (Schiwanes, Schywanes), , Yarrowia genus, Hansenula genus and the like.

When yeast is used as a host cell, the expression vector is usually an origin of replication (if amplification in the host cell is needed) and a selectable marker for growth of the vector in E. coli, induction for recombinant protein expression in yeast. It is preferred to include a sex promoter and terminator, and a selectable marker for yeast.

When the expression vector is a non-integrated vector, it is preferable that the expression vector further contains an autonomously replicating sequence (ARS). This can improve the stability of the expression vector in cells (Myers, AM, et al. (1986) Gene 45:299-310).

Examples of the vector when yeast is used as a host cell include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, PGAPZα, PGAPZα, PGAPZα, B etc. can be mentioned.

Specific examples of the promoter when yeast is used as a host cell include galactose-inducible gal 1 promoter and gal 10 promoter; copper-inducible CUP 1 promoter; thiamine-inducible nmt1 promoter; and methanol-inducible AOX1 promoter, AOX2. Examples thereof include promoters, DHAS promoters, DAS promoters, FDH promoters, FMDH promoters, MOX promoters, ZZA1, PEX5-, PEX8- and PEX14-promoters.

As a method for introducing an expression vector into yeast, any method can be used as long as it is a method for introducing DNA into yeast. For example, electroporation method (Methods Enzymol., 194, 182 (1990)), spheroplast Method (Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)), lithium acetate method (J. Bacteriol., 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

Examples of filamentous fungi include the genus Acremonium, the genus Aspergillus, the genus Ustilago, the genus Trichoderma, the genus Neurospora, the genus Fusarium, and the genus Fusarium. Penicillium genus, Myceliophtora genus, Botrytis genus, Magnaporthe genus, Mucor genus, Metalithium (Metarhizus genus), Monascus (Rhizopus) , And bacteria belonging to the genus Rhizomucor.

Specific examples of the promoter when a filamentous fungus is used as the host cell include salicylic acid-inducible PR1a promoter; cycloheximide-inducible Placc promoter; and quinic acid-inducible Pqa-2 promoter.

The expression vector can be introduced into the filamentous fungus using a conventionally known method. For example, the method of Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)], protoplast method [Mol. Gen. Genet. , 168:111 (1979)], the competent method [J. Mol. Biol. 56:209 (1971)], electroporation method and the like.

The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected depending on the type of host.

A promoter can be any array of DNA sequences that specifically interacts with cellular transcription factors to regulate the transcription of downstream genes. The choice of a particular promoter depends on which cell type is used to express the protein of interest. The transcriptional regulatory sequence can be from the host microorganism. Depending on the host cell, known promoters capable of manipulating the function of the host cell can be used.

Examples of promoters widely used in recombinant technology include, for example, prokaryotic inducible promoters such as trp, recA, lacZ, AraC of major right and left promoters of bacteriophage, and gal promoter of E. coli, α-of Bacillus subtilis. Amylase (Ullmanen Ett at., J. Bacteriol. 162:176-182, 1985) and σ-D specific promoter (Gilman et al., Gene sequence 32: 11-20 (1984)), Bacillus bacteriophage promoter. (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), the promoter of Streptomyces (Ward et at., MoI.-6, 203. Gene. 68: 203). including. Exemplary prokaryotic promoters are Glick (J. Ind. Microtiot. 1:277-282, 1987); Cenatempo (Biochimie 68:505-516, 1986); and Gottesranan (Ann. Rev. Genet. 41:41. 442, 1984).

(Target protein)
The recombinant cell according to one embodiment of the present invention expresses a target protein. The target protein means a protein whose purpose is to recover and utilize the protein after expressing it by the protein expression method. Examples of the target protein include any protein that is preferably produced on an industrial scale, and examples thereof include a protein that can be used industrially, a protein that can be used medically, and a structural protein. Specific examples of proteins that can be used industrially or medically include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used for vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, Examples include allergens, full-length antibodies, antibody fragments or derivatives, and the like. Specific examples of structural proteins include fibroin (eg, spider silk, silkworm silk, etc.), keratin, collagen, elastin, resilin, fragments of these proteins, and proteins derived therefrom.

As used herein, fibroin includes naturally occurring fibroin and modified fibroin. As used herein, "naturally-derived fibroin" means a fibroin having the same amino acid sequence as naturally-derived fibroin, and "modified fibroin" means a 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 spider silk fibroin include spider silk protein produced by spiders.

Fibroin is, 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. May be. 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 typically, but not limited to, regions having no repeat of the amino acid motif characteristic of fibroin, and consist of about 100 amino acids.

In the present specification, the “domain sequence” refers to a crystalline region (typically corresponding to the (A) _n motif of an amino acid sequence) and an amorphous region (typically, a REP of an amino acid sequence) peculiar to fibroin. The amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. Means an array. Here, the (A) n motif represents an amino acid sequence mainly containing an alanine residue, and the number of amino acid residues is 2 to 27. (A) 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 of only alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 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 or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Examples of naturally-occurring fibroin include, 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. The proteins included can be mentioned. Specific examples of naturally-derived fibroin include, for example, fibroin produced by insects or arachnids.

Examples of fibroin produced by insects include Bombyx mori (Bombyx mori), mulberry (Bombyx mandarina), terrestrial silkworm (Antheraea yamai mai), 柞蚓ya pyramy (Atera ea peri pyna), Anteraea peri ny (Atera pera pyna), Pomegranate (Anteraea pernii) ), silk proteins produced by silkworms such as Anthera ea limata (Antheraea assama), and silk proteins produced by Bombyx mori (Antheraea assama), such as silkworms (Samia cynthia), chrysanthemums (Caligura japonica), chusser silkworms (Antheraea mylitta), and moth silkworms (Antherae a assama). Hornet silk protein is mentioned.

More specific examples of fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence) and AAA278840.1 (amino acid sequence)).

Examples of fibroin produced by spiders include spiders belonging to the genus Araneus, such as Onigumo, Niwaonigamo, Akanonigumo, Aonigumo and Maenoonigumo, spiders of the genus Araneus, spiders of the genus Neon sp. Spiders belonging to the genus Proton, spiders belonging to the genus Pronus, spiders belonging to the genus Cyrtarachne such as Torinofundamas and Otorinofundamas; Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius such as Mameitaiseki spider and Mutsugai spider, and belonging to the genus Argiopsis such as Argiogiope, Argiope brue and Argiope spp. Spiders belonging to the genus Arachnura, Spiders belonging to the genus Acusilas such as Spider Spider, spiders belonging to the genus Cytophora (Spider Spider), such as Spiders, Spiders, and Black Spiders (genus Cytophora). Spider silk protein produced by spiders belonging to the genus Cyclopsa (genus Cyclosa), such as spiders, spider silk proteins, and spider silk spiders belonging to the genus Cyclozopes, such as the spider silk spider, and the spider silk spider, which belong to the genus Cyclosopes. Spiders belonging to the genus Tetragnatha, such as the herring-tailed spider, the pearl-billed spider, and the white-bellied spider, the spider belonging to the genus Leucaug Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira, such as black spiders, spiders belonging to the genus Dyschiriognatha, such as dwarf spiders, such as black spiders, black widow spiders, black widow spiders, and black spiders. Spiders produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus (Latrodectus) and spiders belonging to the genus Euprosthenops (Euprosthenops) Examples include pider silk protein. Examples of the spider silk protein 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 Capra hircus type I keratin.

As the collagen-derived protein, for example, a protein containing a domain sequence represented by Formula 3: [REP2]p (wherein, 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 are arbitrary amino acid residues other than Gly. A plurality of REP2s may be the same amino acid sequence or different amino acid sequences. it can.

Examples of the elastin-derived protein include proteins having amino acid sequences such as NCBI GenBank Accession Nos. AAC98395 (human), I47076 (sheep), and NP786966 (bovine).

As the protein derived from resilin, for example, a protein containing a domain sequence represented by Formula 4: [REP3] _q (wherein, _q is an integer of 4 to 300 in Formula 4, REP3 is Ser-JJ). Shows an amino acid sequence composed of -Tyr-Gly-U-Pro, J represents an arbitrary amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr, U is arbitrary. The amino acid residue is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser, and a 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 indices (hydropathic index, HI) of all amino acid residues that compose the target protein was calculated, and then the sum was divided by the total number of amino acid residues (average HI, hereinafter "hydrophobicity"). Is also represented) is preferably -1.0 or more. Regarding the hydrophobic index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic charactor of a pro. 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 is -0.9 or higher, -0.8 or higher, -0.7 or higher, -0.6 or higher, -0.5 or higher, -0.4 or higher. , -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, for example, 600 kDa or less, 500 kDa or less, 400 kDa or less. , 300 kDa or less, or 200 kDa or less. Generally, the larger the molecular weight of a protein, the easier it is for aggregation to occur.

In one embodiment of the invention, expression of the protein of interest may be inducible. For example, by using an inducible promoter that functions in a host cell and is capable of inducing the expression of a target protein as a promoter that controls the expression of the target protein, the expression of the target protein can be inducible. Inducible promoters include the presence of inducers (expression inducers) such as isopropyl-β-thiogalactopyranoside (IPTG), the absence of repressor molecules, or increase or decrease in temperature, osmotic pressure or pH value. It is a promoter that can control transcription by physical factors.

(culture)
The medium for culturing the recombinant cells is not particularly limited, and can be selected from known natural medium or synthetic medium according to the type of the recombinant cells. As the medium, for example, a liquid medium containing a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, and other components selected from various organic components and inorganic components as necessary can be used. Those skilled in the art may appropriately set the types and concentrations of the medium components.

In the method for producing a target protein according to one embodiment of the present invention, the culture can be aerobically carried out, for example, by aeration culture or shaking culture. The culturing can be carried out by a batch culture, a fed-batch culture, a continuous culture, or a combination thereof. The pH of the medium may be, for example, 3.0 to 9.0. The culture temperature is, for example, 15 to 40°C. The culture time may be, for example, 1 to 60 hours.

The culture conditions are not particularly limited as long as the recombinant cells can grow and the target protein can be accumulated in the insoluble fraction in the recombinant cells expressing the target protein. The recombinant cells may or may not grow during the period in which the target protein is being expressed. The culture conditions may or may not be the same in the period before the target protein is expressed and the period after the expression is started. Cultivation conditions can be appropriately set by those skilled in the art according to various conditions such as the type of technique for forming inclusion bodies.

The culture temperature usually has a great influence on the growth of microorganisms. Generally speaking, the lower limit temperature of growth is 0° C., which is the freezing temperature of water in cells, or a temperature slightly lower than it, and the upper limit temperature is determined by the denaturing temperature of high molecular 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 of growth is 0 to 15°C, the upper limit temperature is 46°C, and the optimum growth temperature is around 36 to 42°C. When the microorganisms are classified according to the optimum growth temperature, they are psychrophilic bacteria having an optimum temperature of 20°C or lower, mesophilic bacteria having an optimum temperature of 20 to 45°C, and thermophilic bacteria having an optimum temperature of 45°C or higher. Can be divided. Here, the optimum growth temperature refers to the temperature at which the microorganism to be cultured can obtain the maximum specific growth rate, and the specific growth rate refers to the growth rate per unit amount of the microorganism, which is a value specific to the microorganism. Varies depending on culture conditions.

In one embodiment of the present invention, the "optimum 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 the culture. The temperature that can be achieved. In one embodiment of the present invention, when the recombinant cell expresses the target protein (after the induction of the expression of the target protein when the expression of the target protein is inducible), the growth of the recombinant cell is controlled by adjusting the culture temperature or the like. By cooling or maintaining the recombinant cell at a temperature lower than the optimum temperature, the expression level of the target protein in the recombinant cell can be increased. Further, by cooling or maintaining the recombinant cells at a temperature lower than the optimum growth temperature of the recombinant cells, the expression level of the target protein in the insoluble fraction can be increased. The temperature lower than the optimum growth temperature of the recombinant cell may be, for example, a temperature 3 to 25°C lower than the lower limit of the optimum growth temperature of the recombinant cell, or a temperature lower than 8 to 20°C. Well, the temperature may be 10 to 18°C lower, 12°C to 18°C lower, 14°C to 17°C lower, 3 to 10°C lower, 5 The temperature may be 8°C lower.

(Lower alcohol)
In the method for producing a target protein according to an embodiment of the present invention, recombinant cells expressing the target protein may be cultured in the presence of lower alcohol. For example, the target protein may be continuously cultured in the presence of a lower alcohol from a recombinant cell in which the target protein is not yet expressed, and then the target protein may be expressed. The cells may be cultured, or at the same time when the recombinant cells express the target protein, they may be cultured under a lower alcohol. Before culturing the recombinant cells in the presence of lower alcohol, the recombinant cells may be cultured in the absence of lower alcohol. By proliferating the recombinant cells to a predetermined amount before expressing the target protein by expression induction or the like, the target protein can be produced more efficiently. Further, since the culture may be performed in the presence of a lower alcohol, for example, the medium for culturing the recombinant cells may contain the lower alcohol in advance, or the lower alcohol may be added to the medium during the culture.

Further, when the expression of the target protein is inducible, for example, lower alcohol may be added to the medium before the induction of expression of the target protein, or lower alcohol may be added to the medium at the same time as the induction of expression. The lower alcohol may be added to the medium at about the same time as the expression induction, the lower alcohol may be added to the medium after the expression induction, or the lower alcohol may be added to the medium immediately before or after the expression induction. By culturing the above recombinant cells and adding a lower alcohol at the same time as, immediately before or immediately after the induction of expression, the expression level of the target protein in the recombinant cells can be increased. It should be noted that the expression induction of the target protein can be expressed as "at the time of expression induction" collectively and immediately before or after, and the period from the start of culture to the expression induction is "the period before expression induction", from the expression induction to the end of culture. The period of may be represented as "the period after expression induction". The time immediately before the expression induction may be, for example, 60 minutes, 30 minutes, or 15 minutes before the start of the expression induction, for example. Immediately after expression induction may be, for example, 15 minutes, 30 minutes, or 60 minutes after the start of expression induction. Simultaneously with or immediately before or after expression induction means, for example, 60 minutes before to 60 minutes later, 30 minutes before to 30 minutes later, 15 minutes before to 15 minutes later, and 10 minutes before to 10 minutes before, based on the start of the expression induction. It may be after 5 minutes or 5 minutes before to 5 minutes.

As described above, a promoter requiring an expression inducer can be used as an inducible promoter capable of inducing expression of the target protein, but in this case, lower alcohol is not added as the above expression inducer. .. A promoter using a lower alcohol as an expression inducer as an inducible promoter capable of inducing expression of a target protein may be excluded.

The lower alcohol means a straight or branched chain alcohol having 1 to 5 carbon atoms, and specifically, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, tert-. Examples include butanol, 1-pentanol, 2-pentanol, 3-pentanol, amyl alcohol, neopentyl alcohol and the like. The preferred lower alcohol is methanol or ethanol, and the more preferred lower alcohol is ethanol.

The lower alcohol can be added by one shot or drop-in. Alternatively, it may be added by a shower-like injection method.

The concentration of lower alcohol in the medium may be, for example, 0.1 to 10 w/v%, 1 to 5 w/v%, 5 to 10 w/v%, or 1 to 3 w/v%.

(Insoluble fraction-inclusion body)
The method for producing a target protein includes accumulating the target protein in an insoluble fraction. In the present specification, the bacterial cells collected from the shaking culture solution are treated with Freeze thaw, suspended in 20 mM Tris-HCl buffer (pH 8.0), and a sonicator (ULTRA SONIC HOMOGENIZER UH-50, SMT Company) is used. The microbial cells are disrupted under the condition of OUTPUT 8 for 1 minute×6 times, and the resulting disrupted cell suspension is subjected to centrifugal separation to obtain a supernatant as a soluble fraction and a precipitate after centrifugation as an insoluble fraction.

The insoluble fraction derived from the cytoplasm is composed of cell debris and inclusion bodies. Inclusion bodies mean insoluble aggregates in the cytoplasm. When the expressed protein is hydrophobic or does not fold properly, it aggregates into inclusion bodies. Therefore, the main component of inclusion bodies is protein.

By using the denaturing agent, the target protein can be solubilized from the purified inclusion body. As the denaturant, for example, a guanidine hydrochloric acid solution, a urea solution or the like can be used. Non-target proteins and nucleic acids may be mixed in the inclusion body.

When a target protein having a periplasmic enzyme or a periplasmic transport signal is expressed, localization of the expressed protein to the periplasm is achieved, and inclusion bodies may be formed in the periplasm.

In one embodiment of the present invention, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% with respect to the entire target protein expressed by the recombinant cells (total expression level). It is preferable that at least 99% or more of the target protein is accumulated in the insoluble fraction. Here, for example, the total amount of the soluble fraction and the insoluble fraction can be regarded as the total amount of the target protein. The ratio of the target protein accumulated in the insoluble fraction is analyzed by a method known to those skilled in the art such as SDS-PAGE and Western blotting to quantify the amounts of the soluble fraction and the protein contained in the insoluble fraction. This can be calculated.

It is presumed that almost all the proteins contained in the insoluble fraction form inclusion bodies. Therefore, the ratio of the target protein accumulated in the insoluble fraction to the whole can be regarded as the ratio of the target protein contained in the inclusion body. For example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the entire target protein expressed by the recombinant cells (total expression level) It is preferable that the target protein of (3) is contained in the inclusion body (forming an inclusion body). It is expected that the inclusion body can be used to protect the target protein from digestion by proteases present in the recombinant cells and to easily purify it by disruption of the recombinant cells (eg, bacterial cells) followed by centrifugation.

The method for producing a target protein according to one embodiment of the present invention may further include collecting the target protein accumulated in the insoluble fraction. Recovery of the target protein can be carried out by a general method such as centrifugation, filtration with a filter such as a drum filter or a press filter.

1) Preparation of plasmid expression strain Based on the base sequence and amino acid sequence of MaSp1, which is one of the fibroin proteins derived from Nephila clavata, a modified fibroin having the amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as “PRT967”). Also referred to as "." The amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of Masp1 derived from Nephila clavata. Furthermore, the amino acid sequence shown in SEQ ID NO: 2 (tag sequence and hinge sequence) is added to the N-terminus.

Next, a nucleic acid encoding PRT967 was synthesized. An NdeI site at the 5'end and an EcoRI site downstream of the stop codon were added to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Then, the same nucleic acid was digested with restriction enzymes NdeI and EcoRI and cut out, and then recombined into a protein expression vector pET-22b(+) to obtain an expression vector.

(2) Inducible Expression of Target Protein Escherichia coli BLR(DE3) (ilvAT219C) was transformed with the pET22b(+) expression vector containing the nucleic acid encoding the protein having the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, referred to as this The transformed E. coli is also referred to as "transformed E. coli". The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours and added to the LB medium containing ampicillin so that the OD ₆₀₀ was 0.03. The culture solution temperature was maintained at 37° C., shake culture was performed in a flask until OD ₆₀₀ reached 0.4, and then isopropyl-β-thiogalactopyranoside (IPTG) having a final concentration of 1 mM and a final concentration of 1% or 3% ethanol (final concentration 1% or 3%) was added, and then shaking culture was carried out at 20° C. for 20 hours.

(3) Confirmation of expressed protein After shaking treatment of 2.5 ml of the shaking culture medium after expression induction, the supernatant was removed to collect the bacterial cells. The cells were suspended in 100 μl of a 2M lithium chloride-containing DMSO solution and treated at 80° C. to solubilize the cell proteins. After 1 μl of the treated sample, 4 μl of 7.5 M Urea and 5 μl of SDS sample buffer were added, followed by treatment at 95° C. for 5 minutes to perform SDS-PAGE. Subsequently, His-tag staining was performed using an Invision staining solution to detect the target protein. The result is shown in FIG. The first to third columns from the left show the results of electrophoresis of cell-derived samples cultured at 20° C. after addition of IPTG. -, 1% and 3% indicate the final concentration of EtOH added (- indicates not added). The arrow indicates the position of the band based on the size of the target protein. Table 2 shows the results of quantifying the band intensity of the target protein using ImageJ (version: 2.0.0-rc-68/1.52h). The band intensity of the target protein of each sample added with 1% EtOH (column 2) and the sample added with 3% EtOH (column 3) is shown as a relative value to the band intensity of the target protein of the sample without addition of EtOH (column 1).

The expression of the target protein in each of the soluble fraction and the insoluble fraction was confirmed as follows. 25 ml of the shaken culture solution after the induction of expression was subjected to centrifugation, and the supernatant was removed to collect the bacterial cells. After the Freeze Thaw treatment, the cells were suspended in 1 ml of 20 mM Tris-HCl buffer (pH 8.0), and a sonicator (ULTRA SONIC HOMOGENIZER UH-50, SMT Company) was used for 1 minute x 6 times under the condition of OUTPUT8. The cells were disrupted at. A 100 μl aliquot of the disrupted cell suspension was centrifuged, and the supernatant was aliquoted as a soluble fraction. On the other hand, the precipitate (insoluble fraction) after centrifugation was suspended in 100 µl of a 2M lithium chloride-containing DMSO solution and treated at 80°C. Each solution of the soluble fraction and the insoluble fraction was subjected to SDS-PAGE in the same manner as in FIG. 1, and then the target protein was detected using an Invision staining solution. The result is shown in FIG. Sup (rows 1 to 3) shows a soluble fraction, and Ppt (rows 4 to 6) shows an insoluble fraction. In addition, the 7th column shows the migration results of the protein molecular weight markers. -, 1% and 3% indicate the final concentration of EtOH added (- indicates not added). Further, the arrow indicates the position of the band based on the size of the target protein. Table 3 shows the results of quantifying the band intensity of the target protein in the insoluble fraction sample using ImageJ (version: 2.0.0-rc-68/1.52h). The band intensity of the target protein of each sample with addition of 1% EtOH (column 5) and 3% (column 6) is shown as a relative value to the band intensity of the target protein of the sample without addition of EtOH (column 4).

Claims (10)

A method for producing a target protein, comprising:
A method comprising culturing a recombinant cell expressing a target protein in the presence of a lower alcohol to accumulate the target protein in an insoluble fraction. The method according to claim 1, wherein the expression of the target protein is inducible. The method according to claim 1, wherein 80% or more of the total expression amount of the target protein expressed by the recombinant cell is accumulated in the insoluble fraction. The method according to claim 1, wherein the target protein has a hydrophobicity of −1.0 or more. The method according to any one of claims 1 to 4, which comprises cooling or maintaining the recombinant cell at a temperature lower than the optimum growth temperature of the recombinant cell. The method according to claim 1, wherein the lower alcohol is ethanol. The method according to claim 1, wherein the target protein is a structural protein. The method according to claim 1, wherein the target protein is fibroin. The method according to any one of claims 1 to 8, wherein the target protein is spider silk fibroin. The method according to any one of claims 1 to 9, wherein the recombinant cell is a cell of a bacterium of the genus Escherichia.