WO2019103104A1 - Système d'expression de protéine recombinante, vecteur d'expression, cellules recombinantes et procédé de production de protéine recombinante - Google Patents

Système d'expression de protéine recombinante, vecteur d'expression, cellules recombinantes et procédé de production de protéine recombinante Download PDF

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WO2019103104A1
WO2019103104A1 PCT/JP2018/043201 JP2018043201W WO2019103104A1 WO 2019103104 A1 WO2019103104 A1 WO 2019103104A1 JP 2018043201 W JP2018043201 W JP 2018043201W WO 2019103104 A1 WO2019103104 A1 WO 2019103104A1
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promoter
seq
sequence
amino acid
recombinant
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昂文 野田
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Ｓｐｉｂｅｒ株式会社
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • 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

Definitions

  • the present invention relates to a recombinant protein expression system, an expression vector, a recombinant cell, and a method for producing a recombinant protein. More specifically, the present invention relates to an expression system and the like combining a novel T7-like promoter and a T7-like RNA polymerase.
  • T7 recombinant protein expression system a recombinant protein expression system combining T7 promoter and T7 RNA polymerase (T7 recombinant protein expression system) is used as an expression system that can express and obtain a large amount of recombinant proteins, and is used as a T7 system, pET It can also be obtained from Novagen as a system.
  • Patent Document 1 discloses T7-derived promoter-driven protein expression having an operator sequence downstream of the T7 promoter sequence and having an additional operator sequence upstream of the T7 promoter sequence. A system is disclosed.
  • Patent Document 2 discloses a protein expression system comprising a) T7A1, T7A2, T7A3, ⁇ pL, ⁇ pR, lacUV5, trp, trc, phoA and rrnB An expression system is disclosed comprising a promoter selected from the group; and b) complete palindromic operator sequences.
  • An object of the present invention is to provide an expression system having higher expression efficiency of recombinant protein than conventional expression systems.
  • the present inventors have used a conventional T7 recombinant protein expression system by combining and using a T7-like promoter in which the base sequence of the T7 promoter has been modified, and a T7-like RNA polymerase having a different recognition format of T7 RNA polymerase and promoter. By comparison, it was found that the expression level of recombinant protein can be dramatically improved. The present invention is based on this finding.
  • the present invention provides, for example, the following [1] to [15].
  • a recombinant protein expression system A T7-like promoter comprising a promoter sequence having a sequence identity of 50% or more and 95% or less with the T7 promoter sequence shown in SEQ ID NO: 8;
  • a nucleic acid encoding a recombinant protein under the control of said T7-like promoter A nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence that has at least 40% sequence identity to the amino acid sequence shown by SEQ ID NO: 9.
  • the T7-like promoter has a transcription efficiency when combined with a T7-like RNA polymerase having the amino acid sequence shown in SEQ ID NO: 9, and has the T7 promoter sequence shown in SEQ ID NO: 8 and T7 having the amino acid sequence shown in SEQ ID NO: 9
  • the expression system according to [1] or [2] which is at least twice as high as the transcription efficiency when combined with RNA-like RNA polymerase.
  • T7-like RNA polymerase has an activity of performing transcription by a T7-like promoter containing the base sequence shown in SEQ ID NO: 1.
  • a T7-like promoter comprising a promoter sequence having a sequence identity of 50% or more and 95% or less with the T7 promoter sequence shown in SEQ ID NO: 8; And a nucleic acid encoding a recombinant protein under the control of the T7-like promoter.
  • [6] The expression vector according to [5], further comprising a nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence having at least 40% sequence identity to the amino acid sequence shown by SEQ ID NO: 9.
  • the expression vector according to [5] or [6] which is a plasmid vector or a viral vector.
  • a reaction mixture for recombinant cell or cell-free protein synthesis comprising the expression system according to any one of [1] to [4].
  • [9] A recombinant cell transformed with the expression vector of any one of [5] to [7].
  • the recombinant cell according to [8] or [9] which is a prokaryote.
  • the above-mentioned prokaryote is a microorganism belonging to a genus selected from the group consisting of Escherichia, Vibrio, Brevibacillus, Serratia, Bacillus, Microbacterium, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • the recombinant cell according to [12] wherein the eukaryote is a yeast, filamentous fungus or insect cell.
  • the above yeast is a yeast belonging to a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Clyberomyces, Trichosporon, Ciwaniomyces, Pichia, Candida, Yarowia and Hansenula [13], The recombinant cell described in. [15] A method for producing a recombinant protein using the recombinant cell according to any one of [8] to [14].
  • T7 promoter pT7, sequence number 8
  • T7-like promoter pT7L1, sequence number 1.
  • T7RNAP T7 RNA polymerase
  • T7LRNAP T7 like RNA polymerase
  • T7LRNAP T7 like RNA polymerase
  • the expression system according to this embodiment is a system for expressing a target recombinant protein, which comprises a T7-like promoter, a nucleic acid encoding a recombinant protein under the control of a T7-like promoter, and a T7-like RNA polymerase And a nucleic acid encoding
  • a T7-like promoter refers to a promoter sequence (hereinafter also referred to as "consensus region equivalent sequence") having 50% to 95% sequence identity to the T7 promoter sequence shown in SEQ ID NO: 8 It refers to a nucleotide sequence having promoter activity.
  • the T7-like promoter is a promoter that is recognized by a T7-like RNA polymerase having the amino acid sequence shown in SEQ ID NO: 9 and can be involved in the initiation of transcription of gene sequences downstream thereof, while the amino acid sequence shown in SEQ ID NO: 10 For T7 RNA polymerase which has a promoter that has little or no involvement in the initiation of transcription.
  • the T7-like promoter according to this embodiment has a transcription efficiency when combined with a T7-like RNA polymerase having the amino acid sequence shown in SEQ ID NO: 9, the T7 promoter sequence shown in SEQ ID NO: 8 and the amino acids shown in SEQ ID NO: 9 Compared with the transcription efficiency when combined with a T7-like RNA polymerase having a sequence, it is preferably at least 2 times or more, more preferably 10 times or more, and still more preferably 100 times or more, Even more preferably, it is 400 times or more.
  • the T7-like promoter according to the present embodiment may be a promoter that contains a consensus region equivalent sequence, and a promoter further including a nucleotide sequence that affects promoter activity before or after (upstream and / or downstream) the sequence is not excluded.
  • the consensus region corresponding sequence has a sequence identity of 50% or more and 95% or less with the T7 promoter sequence shown in SEQ ID NO: 8.
  • the T7 promoter sequence set forth in SEQ ID NO: 8 is a consensus region of the T7 promoter that has been utilized in conventional T7 recombinant protein expression systems. Therefore, the consensus region equivalent sequence is modified to replace, delete, insert and / or add one or more residues to the conventional T7 promoter consensus region (T7 promoter sequence shown in SEQ ID NO: 8). It can also be said that
  • sequence identity refers to the percentage (%) of matching residues when base sequences to be compared or amino acid sequences are multiply aligned (multiple alignment).
  • multiple alignment in order to make the base sequences or amino acid sequences comparable to each other, the base sequences are aligned by appropriately inserting gaps (gaps) so that the corresponding base sequences or amino acid sequence parts are aligned. I say something.
  • a known multiple alignment program can be used. For example, Clustal W, Clustal X, BLAST programs and the like can be suitably used.
  • This consensus region of the T7 promoter is important because it affects the efficiency of promoter recognition and transcription by RNA polymerase, but the regions other than the consensus region do not greatly affect the efficiency of promoter recognition and transcription.
  • the nucleotide sequence of the region of (1) need not be identical to the nucleotide sequence of the T7 promoter. Depending on the host used for transformation, the sequence may be modified according to the host at appropriate times.
  • the residue corresponding to the ninth cytosine (C) residue of the T7 promoter sequence shown in SEQ ID NO: 8 is a thymine (T) residue. This results in higher expression efficiency of the recombinant protein when combined with T7-like RNA polymerase.
  • the consensus region corresponding sequence is that the residue corresponding to the sixth cytosine (C) residue of the T7 promoter sequence shown in SEQ ID NO: 8 is an adenine (A) residue, and the eighth adenine (A) A residue corresponding to a base is a thymine (T) residue, a residue corresponding to a tenth thymine (T) residue is a guanine (G) residue, and a 19th guanine ( G) the residue corresponding to the residue is an adenine (A) residue, the residue corresponding to the 20th guanine (G) residue is a thymine (T) residue, and It is more preferable that the residue corresponding to the guanine (G) residue of is an adenine (A) residue, more preferably at least one, and still more preferably all.
  • the consensus region corresponding sequence is a sequence having all of the above-mentioned modifications
  • a T7-like promoter (pT7L1) having the base sequence shown in SEQ ID NO: 1
  • T7-like promoters (pT7L2 to pT7L7) having the nucleotide sequence shown in any of SEQ ID NOs: 2 to 7.
  • T7-like promoter having the base sequence shown in SEQ ID NO: 1
  • T7 promoter having the base sequence shown in SEQ ID NO: 8 is 52% (11/21) (see FIG. 1) .
  • the consensus region equivalent sequence may have 50% to 95% sequence identity with the T7 promoter sequence shown in SEQ ID NO: 8, 50% to 90%, It may be 50% to 85%, and may be 55% to 80%.
  • the consensus region equivalent sequence may be 60% or more and 65% or more in sequence identity with the T7-like promoter (pT7L1) shown in SEQ ID NO: 1 It may be 70% or more, 80% or more, 85% or more, or 90% or more.
  • T7-like RNA polymerase (hereinafter sometimes referred to as "T7LRNAP”) is an RNA polymerase having an amino acid sequence having at least 40% sequence identity to the amino acid sequence shown in SEQ ID NO: 9
  • T7-like RNA polymerase is an RNA polymerase having an activity of performing transcription by a T7-like promoter shown in SEQ ID NO: 1.
  • RNA polymerase having the amino acid sequence set forth in SEQ ID NO: 9 can be mentioned.
  • FIG. 2 shows multiple alignment of the amino acid sequences of T7 RNA polymerase (T7 RNAP, SEQ ID NO: 10) used in the conventional T7 recombinant protein expression system and T7-like RNA polymerase (T7 LRNAP, SEQ ID NO: 9) according to one embodiment. It is a schematic diagram which shows a result. The sequence identity of T7 RNAP and T7-like RNAP was 33%, and the similarity was 48%.
  • the sequence identity with the amino acid sequence shown by SEQ ID NO: 9 is preferably 60% or more, more preferably 70% or more, and 80% or more. Some are more preferable, 85% or more is still more preferable, 90% or more is still more preferable, and 95% or more is particularly preferable.
  • the recombinant protein produced by the expression system according to the present embodiment may be any protein as long as it is a protein to be obtained, but an industrial scale Those intended for the preparation of For example, proteins usable for industrial use, proteins usable for medical use, structural proteins and the like can be mentioned. Specific examples of proteins that can be used for industrial or medical use include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used for vaccination, vaccines, antigen binding proteins, immunostimulatory proteins, Mention may be made of allergens, full-length antibodies or antibody fragments or derivatives. Specific examples of structural proteins include spider silk, silkworm silk, keratin, collagen, elastin and resilin, and proteins derived therefrom.
  • a protein comprising a domain sequence represented by Formula 1: [(A) n Motif-REP] m (wherein, in Formula 1, (A) The n motif indicates an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 80% or more.
  • An amino acid sequence composed of groups is shown, m is an integer of 8 to 300.
  • a plurality of (A) n motifs may be identical amino acid sequences to each other or may be different amino acid sequences. The amino acid sequences may be identical or different from each other.
  • a protein comprising the amino acid sequence shown in SEQ ID NO: 11 PRT410
  • a protein derived from collagen for example, a protein comprising a domain sequence represented by the formula 2: [REP2] o (wherein, in the formula 2, o represents an integer of 5 to 300.
  • REP2 represents Gly-X-Y
  • X and Y each represent any amino acid residue other than Gly, and a plurality of REP2 may be identical amino acid sequences to each other or may be different amino acid sequences).
  • SEQ ID NO: 99 a protein comprising the amino acid sequence shown by SEQ ID NO: 99 can be mentioned.
  • the amino acid sequence shown in SEQ ID NO: 99 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (Genebank Accession Nos .: CAA56335.1, GI: 3702452) obtained from the NCBI database.
  • the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 100 is added to the N-terminus of the amino acid sequence from residue 301 to residue 540.
  • REP3 As a protein derived from resilin, for example, a protein containing a domain sequence represented by the formula 3: [REP3] p (wherein, in the formula 3, p represents an integer of 4 to 300.
  • REP3 is Ser-J-J-J-J
  • An amino acid sequence consisting of Tyr-Gly-U-Pro is shown, J is any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr U is any one.
  • the amino acid residue is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser.
  • the plurality of REP3 present may be the same or different amino acid sequences. Can be mentioned.
  • a protein comprising the amino acid sequence shown in SEQ ID NO: 101 can be mentioned.
  • Thr at position 87 is substituted with Ser in the amino acid sequence of resilin (gene accession numbers NP 611 157, Gl: 24654243 of NCBI), and Asn at position 95
  • the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 100 is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which
  • a protein derived from elastin for example, a protein having an amino acid sequence of Accession Nos. AAC98395 (human), I47076 (sheep), NP786966 (bovine) of Genebank of NCBl can be mentioned.
  • a protein comprising the amino acid sequence shown in SEQ ID NO: 102 can be mentioned.
  • the amino acid sequence set forth in SEQ ID NO: 102 is the amino acid sequence set forth in SEQ ID NO: 100 at the N-terminus of the amino acid sequence from residues 121 to 390 of the amino acid sequence of Accession No. AAC98395 of Genebank of NCBI (tag sequence And hinge arrangement) are added.
  • a protein derived from keratin for example, type I keratin of Capra hircus etc. can be mentioned. Specifically, a protein comprising the amino acid sequence shown in SEQ ID NO: 103 (the amino acid sequence of Accession No. ACY30466 of Genebank of NCB1) can be mentioned.
  • the expression system comprises at least (i) a T7-like promoter, (ii) a nucleic acid encoding a recombinant protein under the control of a T7-like promoter, and (iii) a nucleic acid encoding a T7-like RNA polymerase
  • the specific aspect is not particularly limited.
  • all of (i) to (iii) may be integrated into the genome of the host, and at least one of (i) to (iii) is integrated into the genome of the host, and the others are extrachromosomal DNA (eg, It may be held by the host as a plasmid), and all of (i) to (iii) may be held by the host as an extrachromosomal DNA (eg, a plasmid).
  • the nucleic acid encoding the recombinant protein is under the control of a T7-like promoter means that the transcription of the nucleic acid is performed by the function of the T7-like promoter.
  • the expression system according to this embodiment functions as an expression vector. That is, the expression vector according to the present embodiment comprises (i) a T7-like promoter comprising a promoter sequence having 50% to 95% sequence identity with the T7 promoter sequence shown in SEQ ID NO: 8; (ii) T7 And at least a nucleic acid encoding a recombinant protein under the control of the promoter.
  • the expression vector according to the present embodiment further comprises (iii) a nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence whose sequence identity to the amino acid sequence shown by SEQ ID NO: 9 is 40% or more. It is also good.
  • the expression vector according to the present embodiment does not contain a nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence whose sequence identity with the amino acid sequence shown by SEQ ID NO: 9 is (iii) 40% or more
  • the nucleic acid of (iii) is previously introduced as genomic or extrachromosomal DNA, and combined with a host that expresses T7-like RNA polymerase, it can be used as an expression system according to the present invention.
  • the expression vector according to this embodiment may further comprise (i) one or more regulatory sequences operably linked to a T7-like promoter.
  • regulatory sequences include, for example, sequences that control the expression of recombinant proteins in the host (for example, enhancers, ribosome binding sequences, terminators (transcription termination sequences), etc.), and these may be selected appropriately according to the type of host. It can be selected.
  • any vector capable of autonomous replication in the host cell or integration into the host cell genomic DNA can be used.
  • plasmid vector, virus vector, cosmid vector, fosmid vector And artificial chromosome vectors can be appropriately selected depending on the host cell.
  • expression vectors include pBTrp2, pBTac1 and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by Promega) QIAGEN, pKYP10 (JP-A-58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad.
  • the expression vector according to the present invention further contains a selection marker gene for selection of transformant recombinant cells.
  • a selection marker gene for selection of transformant recombinant cells.
  • resistance genes against various drugs such as tetracycline, ampicillin, kanamycin and the like can be used as a selection marker gene.
  • Recessive selection markers that can complement gene mutations involved in auxotrophy can also be used.
  • eukaryotes for example, yeast
  • a resistance gene against geneticin can be used as a selection marker gene
  • a selection marker such as LEU2, URA3, TRP1, HIS3, etc. is also used it can.
  • filamentous fungi niaD (Biosci.
  • the recombinant cell according to the present embodiment is a recombinant cell containing the expression system according to the present embodiment.
  • the recombinant cell according to the present embodiment can be obtained, for example, by transforming a host with the expression vector according to the present embodiment.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.
  • prokaryotic hosts such as bacteria include microorganisms belonging to the genus Escherichia, Vibrio, Brevibacillus, Serratia, Bacillus, Microbacterium, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. Can.
  • microorganisms belonging to the genus Escherichia include Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BL21 (Merck Millipore), Escherichia coli BLR (DE3) (Merck) Millipore), E. coli DH1, E. coli GI698, E. coli HB101, E. coli JM109, E. coli K5 (ATCC 23506), E. coli KY3276, E. coli MC 1000, E. coli 471 E. coli No.). 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, E.
  • E. coli Tuner Novagen
  • E. coli Tuner DE3 (Novagen)
  • E. coli W1485 E. coli W3110 (E. coli) ATCC 27325)
  • Escherichia coli XL1-Blue Escherichia coli XL2-Blue, and the like.
  • Vibrio avalonicus (V. abalonicus) ATCC 27390, Vibrio adatus (V. adaptatus) ATCC 19263, Vibrio aerogenes (V. aerogenes) ATCC 700797, Vibrio ⁇ Asestrianus (V. aestuarianus) ATCC 35048, Vibrio alginolyticus (V. alginolyticus) ATCC 14582, Vibrio alagosus (V. algosus) ATCC 14390, Vibrio angiillarum (V.
  • Vibrio avalonicus (V. abalonicus) ATCC 27390, Vibrio adatus (V. adaptatus) ATCC 19263, Vibrio aerogenes (V. aerogenes) ATCC 700797, Vibrio ⁇ Asestrianus (V. aestuarianus) ATCC 35048, Vibrio alginolyticus (V. alginolytic
  • anguillalum ATCC 43305, Vibrio carviensis (V. BAA 606, V. campbellii ATCC 25920, V. carchariae ATCC 35084, V. coralliilyticus (V. coralliilyticus) ATCC BAA-450, V. costicola (V. costicola) ATCC 43147, Vibrio chirotrik (V. cyclitrophicus) ATCC 700982, V. cyclosites (ATCC 14635), V. diazotrophicus (V. diazotrophicus) ATCC 33466, V. fischeri (V. fischeri) ATCC 25918, V. biogazogenes (V. Gazogenes) ATCC 29988, V.
  • V. halioticoli (V. halioticoli) ATCC 700,680, V. harveyi (V. harveyi), ATCC 14126, V. hispanica (V. hispanica), ATCC 51589, V. ichthyoenteri (ATC 7010), Piscarius (V. liiopiscarius) ATCC 51760, Vibrio lentus (V. lentus) ATCC BAA-539, Vibrio licufaciens (V. liquefaciens) ATCC 17058, Vibrio rogei (V. logei) ATCC 15382, Vibrio marinagalis (V. (Marinagilis) ATCC 14398, V.
  • pectinicida (V. pectinicida) ATCC 700783 Pelagius) ATCC 33504, Vibrio penaeicida (V. penaeicida) ATCC 51841, Vibrio ponticus (V. ponticus) ATCC 14391, Vibrio proteolyticus (V. proteolyticus) ATCC 53559, Vibrio schloer étalitas (V. psychro rythrus) ATCC 27364, Vibrio salmonicida (V. salmonicida) ATCC 43839, Vibrio syloii (V. shiloii) ATCC BAA-91, Vibrio spectacularus (V.
  • Brevibacillus agri As microorganisms belonging to the genus Brevibacillus, for example, Brevibacillus agri, Brevibacillus bollsterensis, Brevibacillus centropolis Brevibacillus formosas, Brevibacillus imbocatus, Brevibacillus latyrosporus, Brevibacillus limnophilus, Brevibacillus parabrevis Brevibacillus reusseri, Brevibacillus samorvar, Brevibacillus brevis 47 (FERM BP-1223), Brevibacillus brevis 47K (FERM BP-2308), Brevibacillus brevis 47-5 (FERM BP-1664), Brevi Bacillus brevis 47-5Q (JCM8975), Brevibacillus choshinensis HPD 31 (FERM BP-1087), Brevibacil Choshinensis HPD31-S (FERM BP-6623), Brevibac
  • Serratia As a microorganism belonging to the genus Serratia, for example, Serratia liquefacience (ATC 14460), Serratia entomophila (Serratia entomophila), Serratia ficaria (Serratia ficaria), Serratia fonticola (Serratia fonticola), Serratia grimesi (Serratia grimesii), Serratia proteamaculans (Serratia proteamaculans), Serratia odorifera (Serratia odorifera), Serratia plymuthica, Serratia rubidaea etc. It is possible.
  • microorganism belonging to the genus Bacillus examples include, for example, Bacillus subtilis, Bacillus amyloliquefaciens, and the like.
  • microorganism belonging to the genus Microbacterium examples include Microbacterium ammoniafilum ATCC 15354 and the like.
  • Brevibacterium divivalatam (Corynebacterium glutamicum) ATCC 14020, Brevibacterium flavum (Corynebacterium glutamicum ATCC 14067) ATCC 13826, ATCC 14067, Brevibacterium inmariofilum (Brevibacterium immariophilum) ATCC 14068, Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC 13869) ATCC 13665, ATCC 13869, Brevibacterium roseum ATCC 13825, Brevibacterium saccharolyticum TCC14066, Brevibacterium Chiogenitarisu ATCC19240, Brevibacterium album ATCC15111, mention may be made of Brevibacterium Serinumu ATCC15112 and the like.
  • Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC 6871, ATCC 6872, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 14067, Corynebacterium acetoacidophilus Lam (Corynebacterium acetoacidophilum) ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium alkanolitica cam ATCC 21511, Corynebacterium carnae ATCC 15991, Corynebacterium glutamicum Arm ATCC13020, ATCC13032, ATCC13060, can be named Corynebacterium Lilium ATCC15990, Corynebacterium Merasekora ATCC 17965, Corynebacterium thermo amino monocytogenes AJ12340 (FERMBP-1539), Corynebacterium Hakyurisu ATCC13868 and the
  • Pseudomonas As a microorganism belonging to the genus Pseudomonas, for example, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicaerum, Pseudomonas fulva (Pseudomonas sp.) D-0110 etc. can be mentioned.
  • Eukaryotic hosts can include, for example, yeast and filamentous fungi (molds and the like).
  • yeast examples include, for example, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Schwanniomyces, the genus Pichia, and the genus Candida. And yeast belonging to the genus Jalowia and Hansenula.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Schizosaccharomyces pombe Schizosaccharomyces pombe
  • Kleberomyces lactis Kleberomyces marxiplaneculus leutricholine Albius (Schwanniomyces alluvius)
  • Ciwaniomyces occidentalis Candida utilis (Candida utilis)
  • Pichia pastoris ichia pastoris
  • Pichia angusta Pichia
  • Pichia methanolica Pichia polymorpha
  • Pichia stipitis Yarrowia lipolytica (Harrowia lipolytica), Etc.
  • filamentous fungi examples include, for example, Acremonium, Aspergillus, Ustilago, Trichoderma, Neurospora, Fusarium, Fusarium, Humicola, Penicillium (Penicillium), Myceliophtora, Myceliophtora, Botryts (Botryts), Magnaporthe (Magnaporthe), Mucor (Mucor), Metalithium (Metarhizium), Monascus (Monascus), Rhizopus And bacteria belonging to the genus Rhizome core.
  • filamentous fungi include Acremonium alabamense, Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus millorisu Aspergillus oryzae, Aspergillus sake, Aspergillus sojae (Aspergillus sojae), Aspergillus tubigensis, Aspergillus niger (Aspergil niger) us niger), Aspergillus nidulans, Aspergillus parasiticus, Aspergillus ficuum, Aspergillus ficuum, Aspergillus phenicus (Aspergillus phoeicus), Aspergillus , Aspergillus flavus, Aspergillus fumigatus, Aspergillus japonicus (Aspergillus japonicus), Trichoderma vibe De (Trichoderma viride), Trichoderma harzianum, Trichoderma
  • penicillium chrysogenum P. n. (Penicillium canescens), Penicillium emersonii, Penicillium funiculosum, Penicillium gliceolozeum (Penillium canescens) illium griserooseum), Penicillium perpurogenum (Penicillium purpurogenum), Penicillium roqueforuchi (Penicillium roqueforti), Myseriophtra thermofilm (Myceliophtaora thermophilum), Mucoa ambigus (Mucor ambiguus), Mucor fragilis, Mucor hiemalis, Mucor inaequisporus, Mucor oblongiellipticus (Mucor oblongiellipti) us), Mucor racemosus, Mucor recurvus, Mucor saturninus, Mucor subtilissmus, Ogataea polymorpha, Funero cioce
  • insect cells include, for example, insect cells of lepidopterans, and more specifically, insect cells derived from Spodoptera frugiperda such as Sf9 and Sf21, and Trichoplusia such as High 5 and the like.
  • ni include insect cells and the like.
  • any method of introducing DNA into the host cell can be used.
  • the method of Cohen et al. Calcium chloride method [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • protoplast method Japanese Patent Application Laid-Open No. 63-248394, Gene, 17, 107 (1982), method described in Molecular & General Genetics, 168, 111 (1979)
  • spheroplast method Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)
  • Takahashi et al. J. Bacteriol., 1983, 156: 1130-1134
  • the expression system or the expression vector containing the expression system according to the present embodiment may be integrated into a chromosome (chromosomal DNA).
  • a known method can be used as a method of integrating the expression system or the expression vector including the expression system according to the present embodiment into the chromosome of the host, and for example, the recombination mechanism in double strand break repair of ⁇ phage is applied
  • the ⁇ red method, the Red / ET homologous recombination method, and the transfer method using transposon activity using pUT-mini Tn5 can be mentioned.
  • it can be integrated into the host chromosome according to the method described in the kit, using “Transposon-based gene transfer kit with the Transposon: pUTmini-Tn5 Kit” or the like by Biomedal.
  • Selection of recombinant cells transformed with the expression vector according to the present invention can be carried out by plaque hybridization, colony hybridization and the like using a probe that selectively binds to the nucleic acid contained in the expression vector.
  • a probe that selectively binds to the nucleic acid contained in the expression vector.
  • the said probe what modified the partial DNA fragment amplified by PCR method with a radioisotope or digoxigenin based on the base sequence information of the expression vector which concerns on this invention can be used.
  • the target protein can be produced by expressing the nucleic acid encoding the target protein in a recombinant cell containing the expression system according to the present invention.
  • the expression method in addition to direct expression, secretory production, fusion protein expression and the like can be performed according to the method described in Molecular and Cloning 2nd Edition.
  • the target protein when it is expressed by yeast, animal cells or insect cells, the target protein can be obtained as a sugar or glycosylated polypeptide.
  • the target protein is produced, for example, by culturing a recombinant cell containing the expression system according to the present invention in a culture medium, producing and accumulating the target protein in the culture medium, and collecting it from the culture medium.
  • the method for culturing a recombinant cell according to the present invention in a culture medium can be carried out according to a method usually used for culturing a host.
  • the host When the host is a prokaryote such as E. coli or a eukaryote such as yeast, it contains a carbon source, nitrogen source, inorganic salts and the like that can be used by the recombinant cell as a culture medium for the recombinant cell according to the present invention.
  • a prokaryote such as E. coli or a eukaryote such as yeast
  • a carbon source, nitrogen source, inorganic salts and the like that can be used by the recombinant cell as a culture medium for the recombinant cell according to the present invention.
  • a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the recombinant cells.
  • the carbon source may be any as long as the recombinant cells can be utilized, for example, glucose, fructose, sucrose and molasses containing them, carbohydrates such as starch and starch hydrolysate, acetic acid and propionic acid etc. Organic acids and alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include, for example, ammonium, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate
  • other nitrogen-containing compounds such as peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • a medium with high salt concentration for example, a medium containing 0.5 to 5% NaCl is preferable.
  • the culture of prokaryote or eukaryote can be performed under aerobic conditions such as shake culture or submerged aeration culture, for example.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. Adjustment of the pH of the culture medium can be carried out using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia and the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during culture.
  • an inducer may be added to the medium as needed.
  • culturing a recombinant cell transformed with a vector using a lac promoter for expression of T7-like RNA polymerase isopropyl-.beta.-D-thiogalactopyranoside etc. may be displayed with an expression vector using a trp promoter.
  • indole acrylic acid or the like may be added to the medium.
  • TNM-FH medium manufactured by Pharmingen
  • Sf-900 II SFM medium manufactured by Life Technologies
  • ExCell 400 ExCell 405
  • ExCell 405 all manufactured by JRH Biosciences
  • Grace ' s Insect Medium Nature, 195, 788 (1962)
  • Culturing of insect cells can be carried out, for example, for 1 to 5 days under the conditions of pH 6 to 7 of culture medium, culture temperature of 25 to 30.degree.
  • antibiotics such as gentamicin may be added to the culture medium as needed during culture.
  • the protein of interest when the protein of interest is produced in recombinant cells or on a recombinant extracellular membrane, the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Rowe et al. (Proc) Natl.Acad.Sci.USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)), or JP-A-5-336963, WO 94/23021, etc.
  • the target protein can be changed to be actively secreted out of the recombinant cells.
  • the target protein produced by the recombinant cell containing the expression system according to the present invention can be isolated and purified by methods commonly used for isolation and purification of protein. For example, when the protein of interest is expressed in solution in cells, after completion of culture, recombinant cells are recovered by centrifugation, suspended in aqueous buffer, and then sonicated, French press Then, the recombinant cells are disrupted with a Manton Gaulin homogenizer, Dynomil, etc. to obtain a cell-free extract.
  • resin such as diethylaminoethyl (DEAE) -s
  • the recombinant cells are similarly recovered and then disrupted and centrifuged to perform insoluble of the target protein as a precipitate fraction. Recover the body.
  • the insoluble form of the target protein recovered can be solubilized with a protein denaturant.
  • a purified preparation of the target protein can be obtained by the same isolation and purification method as described above.
  • the target protein or its derivative can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the reaction mixture for cell-free protein synthesis according to the present embodiment includes the expression system according to the present embodiment.
  • the reaction mixture for cell-free protein synthesis according to this embodiment can be prepared, for example, as described in Methods in molecular biology, vol. 267, pp. 169-182, Mol. Syst. Biol. , 2008, vol. 4, Article No. 220 and Biotechnol. Bioeng. , 2011, vol. 108, pp.
  • the target protein can be synthesized by preparing according to the known method described in 1570-1578. With respect to the components of the reaction mixture, reference is made, for example, to the descriptions of US Pat. Nos. 7,338,789 and 7,351,563, and US Patent Application Publication Nos. 2010/0184135 and 2010/0093024. be able to.
  • DNA encoding a target protein can be used as a template nucleic acid.
  • the DNA fragments (1) to (3) prepared in the above (i) to (iii) are ligated using the In-Fusion (registered trademark) cloning system (Takara Bio Inc.) to prepare a vector (1). did.
  • the vector (1) was digested with restriction enzymes BglII and SacI and the resulting fragments were linearized, and ligated by Overlap extension PCR (OE-PCR) method using four types of primers shown in Table 4 (see Table 4).
  • OE-PCR Overlap extension PCR
  • An expression vector obtained by ligating the amplified and prepared T7 promoter-containing fragment (see FIG. 3) using In-Fusion (registered trademark) cloning system (Takara Bio Inc.) to remove pelB signal sequence p15A-pT7 / T7 RNAP was constructed.
  • the base sequence of the expression vector p15A-pT7 / T7RNAP is shown in SEQ ID NO: 22. The said base sequence was confirmed by the Sanger method.
  • Expression Vector p15A-HGFP-pT7 / T7RNAP> A DNA fragment (4) containing a sequence encoding Holly green fluorescent protein (HGFP-His) with 6 ⁇ His tag added at the C-terminus using T5-HollyGFP (Cosmo Bio) as a template is shown in Table 5 2 Amplified by PCR using different types of primers (see Table 5). PCR was performed for 35 cycles of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 10 seconds.
  • a fragment obtained by cleaving the expression vector p15A-pT7 / T7RNAP with restriction enzymes NdeI and SacI, and the DNA fragment (4) are ligated using the In-Fusion (registered trademark) cloning system (Takara Bio Inc.) for expression
  • the vector p15A-HGFP-pT7 / T7RNAP (see FIG. 4) was constructed.
  • the nucleotide sequence of HGFP in the expression vector p15A-HGFP-pT7 / T7RNAP was confirmed by the Sanger method.
  • T7-like RNA polymerase (T7LRNAP) gene A DNA fragment (5) containing a nucleotide sequence encoding T7LRNAP having the amino acid sequence set forth in SEQ ID NO: 9 was synthesized by the following method.
  • each primer is designed so that the sense strand and the antisense strand overlap by about 20 bases alternately.
  • a DNA fragment containing a nucleotide sequence encoding T7LRNAP by linking all the primers according to the method shown below by Dual asymmetric PCR (DA-PCR) method using the synthesized primers (T7LRNAP- # 1 to # 66) etc. I got (5).
  • DA-PCR Dual asymmetric PCR
  • the resulting reaction product (a fragment to which the primer added to the reaction tube was specifically linked) was prepared according to the attached manual using Wizard® SV Gel and PCR Clean-Up System (Promega). Refined.
  • Step 2 the purified reaction product (a ligated fragment of T7LRNAP- # 1 to # 8, a ligated fragment of T7LRNAP- # 9 to # 16, a ligated fragment of T7LRNAP- # 17 to # 24, and T7LRNAP- 5 ⁇ L each of # 25 to # 32 ligated fragments) was added to a new reaction tube, and 1 ⁇ L each of Primer RNA P_F (10 pmol / ⁇ L) and Primer T7LRNAP- # 32 (10 pmol / ⁇ L) shown in Table 8 and PrimeSTAR ( 30 ⁇ L of a registered trademark Max (manufactured by Takara Bio Inc.) and ultrapure water were added to a final volume of 60 ⁇ L, and PCR was performed under the same conditions as in the first step DA-PCR.
  • the purified reaction product a ligated fragment of T7LRNAP- # 1 to # 8, a ligated fragment of T7LRNAP- # 9 to # 16, a lig
  • purified reaction products (ligated fragments of T7LRNAP- # 33- # 40, ligated fragments of T7LRNAP- # 41- # 48, ligated fragments of T7LRNAP- # 49- # 56, T7LRNAP- 5 ⁇ L each of the ligated fragments # 57 to # 61 and the ligated fragments of T7 LRNAP- # 62 to # 66) are added to another new reaction tube, and the primers RNA P_R (10 pmol / ⁇ L) shown in Table 8 are further added.
  • the obtained reaction product (a fragment to which the reaction product of the first step added to the reaction tube is specifically linked) was prepared using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega Corp.) And purified according to the attached manual.
  • the obtained ligated fragment (double-stranded DNA fragment containing a nucleotide sequence encoding T7LRNAP) was amplified by PCR using primers T7LRNAP- # 1 and T7LRNAP- # 66 after the nucleotide sequence was confirmed by Sanger method .
  • PCR was carried out for 35 cycles of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 40 seconds.
  • the reaction product obtained by PCR is purified according to the attached manual using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega Corp.), and a DNA fragment containing a nucleotide sequence encoding T7LRNAP ( I got 5).
  • Expression Vector p15A-HGFP-pT7 / T7LRNAP Construction of Expression Vector p15A-HGFP-pT7 / T7LRNAP>
  • an expression vector p15A-HGFP-pT7 / T7LRNAP was constructed in which the nucleotide sequence encoding the T7 RNAP gene in the expression vector p15A-HGFP-pT7 / T7 RNAP was replaced with the nucleotide sequence encoding the T7LRNAP gene.
  • the expression vector p15A-HGFP-pT7 / T7RNAP was amplified by PCR using two types of primers shown in Table 9 (see Table 9) and linearized. PCR was performed for 35 cycles of conditions of 98 ° C. for 10 seconds, 60 ° C. for 5 seconds, and 72 ° C. for 40 seconds.
  • FIG. 5 is a view showing the nucleotide sequence of DNA fragment (6).
  • an expression vector p15A-HGFP-pT7L1 / T7LRNAP was constructed by replacing the T7 promoter sequence in the expression vector p15A-HGFP-pT7 / T7LRNAP with a T7-like promoter (pT7L1) sequence by the following method.
  • the expression vector p15A-HGFP-pT7 / T7LRNAP was cleaved with restriction enzymes BglII and SacI for linearization.
  • the DNA fragment (6) and the linearized p15A-HGFP-pT7 / T7LRNAP fragment were ligated using In-Fusion (registered trademark) cloning system (manufactured by Takara Bio Inc.), and the expression vector p15A was used.
  • -HGFP-pT7L1 / T7LRNAP was obtained.
  • the nucleotide sequence of the obtained expression vector was confirmed by the Sanger method.
  • T7 promoter sequences in the expression vector p15A-HGFP-pT7L2 to pT7L7 / T7LRNAP are shown in Table 11 by the same procedure as for the expression vector p15A-HGFP-pT7L1 / T7LRNAP except that the nucleotide sequences of the primers used were changed.
  • Expression vectors p15A-HGFP pT7L2-pT7L7 / T7LRNAP were constructed in which T7-like promoter (pT7L2-pT7L7) sequences were replaced. The nucleotide sequence of the obtained expression vector was confirmed by the Sanger method.
  • HGFP-expressing recombinant cells Preparation of HGFP-expressing recombinant cells and expression analysis of HGFP> E. coli BL21 (Merck Millipore) and Vibrio natriegens NBRC 15636 were respectively transformed with the two expression vectors p15A-HGFP-pT7 / T7RNAP and p15A-HGFP-pT7L1 / T7LRNAP prepared above. HGFP expressing recombinant cells were generated.
  • the prepared recombinant cells were cultured by the following method to analyze the expression level of HGFP.
  • HGFP-expressing recombinant cells transformed with E. coli BL21 strain 2 mL of LB medium containing 25 ⁇ g / mL chloramphenicol was cultured at 37 ° C. with shaking.
  • HGFP-expressing recombinant cells transformed with Vibrio natriegens NBRC 15636 strain 12.5 ⁇ g / mL chloramphenicol and 11.9 g / L NaCl, 4.7 g / L MgCl 2 ⁇ 6H 2 O and 0.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the expression amount of each HGFP-expressing recombinant cell when the expression amount (average fluorescence intensity) of HGFP-expressing recombinant cells transformed the BL21 strain with the conventional expression system p15A-HGFP-pT7 / T7RNAP is assumed to be 100 ( The relative values of the average fluorescence intensity) are shown in Table 12.
  • T7-like promoter (pT7L1), which is the expression system of the present invention, compared to the expression vector p15A-HGFP-pT7 / T7RNAP composed of T7 promoter (pT7) and T7 RNA polymerase (T7RNAP), which corresponds to the conventional expression system
  • T7LRNAP T7 RNA polymerase
  • T7LRNAP T7-like RNA polymerase
  • E. coli BL21 strain increased about 20 times
  • Vibrio natriegens NBRC 15636 strain also increased about 1.9 times. It has been confirmed that the expression system of the present invention is applicable beyond the genus of the host.
  • E. coli JM109 strain (Takara Bio stock) is a total of 7 types of expression vectors p15A-HGFP-pT7 / T7RNAP, and expression vectors p15A-HGFP-pT7L1 to pT7L7 / T7LRNAP having 7 types of T7-like promoters.
  • the company made product No. 9052) and transformed and cultured, and the expression amount of HGFP was compared.
  • the relative value of the expression level (average fluorescence intensity) of is shown in Table 13.
  • the expression level was increased about 15-fold by substituting AT residues for the 19th and 20th GG residues of pT7 (in particular, comparison between pT7 and pT7L2). Furthermore, it was confirmed that the expression level increased about 20-fold by replacing the 21st G residue of pT7 with the A residue (in particular, comparison between pT7 and pT7L1).
  • E. coli JM109 was similarly transformed and cultured with three types of expression vectors p15A-HGFP-pT7 / T7RNAP, p15AHGFP-pT7 / T7LRNAP and p15A-HGFP-pT7L1 / T7RNAP to express HGFP
  • the compatibility between the promoter and the RNA polymerase was confirmed.
  • the relative value of the expression level (average fluorescence intensity) of is shown in Table 14.
  • the T7-like promoter in the expression system of the present invention is not suitable for T7 RNA polymerase, and in the expression vector p15A-HGFP-pT7L1 / T7RNAP combining these, the expression amount of HGFP is extremely low at 13 (about 0.1 times) It was a thing.
  • the T7 promoter is not suitable for T7-like RNA polymerase, and in the expression vector p15A-HGFP-pT7 / T7LRNAP combining them, the expression level of HGFP is 41 (about 0.4 times), compared to the conventional T7 system. It has fallen to less than half.
  • SSP spider silk protein
  • E. coli BL21 strain was transformed with these expression vectors, and the resulting transformant was cultured and induced for expression by addition of IPTG under the same conditions as described above.
  • expression induction (culture time after IPTG addition) was 6 hours.
  • 100 ⁇ L of culture fluid is collected and centrifuged to remove the supernatant, and the precipitated cells are suspended in 100 ⁇ L of 1 ⁇ Laemmlli buffer, and the suspension is heat-treated at 95 ° C. for 5 minutes Did. Thereafter, the number of cells per volume between samples was equalized by addition of 1 ⁇ Laemmlli buffer. Dilution series (1-fold, 2-fold and 3-fold dilutions) of each sample were prepared and subjected to SDS-PAGE.
  • Spider silk protein was confirmed by InVision (trademark) His-Tag In-Gel Staining Kit (Thermo Fisher Scientific, product number LC6033) which specifically detects His tag.
  • the intensities of the bands detected at the expected molecular weight were quantified by ImageJ (NIH).
  • the slope of the calibration curve was determined from the band intensities of the dilution series.
  • the correlation coefficient R 2 of the calibration curve of each sample was 0.99 or more.
  • the relative value of the slope between samples was taken as the relative value of the expression level between samples.
  • Expression vector p15A-SSP-pT7L1 which is the expression system of the present invention, where the expression amount of the strain obtained by transforming BL21 strain with expression vector p15A-SSP-pT7 / T7RNAP is 100, which corresponds to the conventional expression system
  • the relative expression levels of the strain obtained by transforming strain BL21 with / T7LRNAP are shown in Table 15.
  • expression vector p15A-SSP-pT7L1 / T7LRNAP composed of a T7-like promoter and a T7-like RNA polymerase like HGFP.
  • the expression amount was about 5 times that of the conventional expression system (expression vector p15A-SSP-pT7 / T7RNAP).

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Abstract

La présente invention concerne un système d'expression de protéine recombinante comprenant : un promoteur de type T7 qui comprend une séquence de promoteur présentant une identité de séquence de 50 à 95 % par rapport à une séquence de promoteur T7 représentée par la séquence SEQ ID NO:8 ; un acide nucléique qui code pour une protéine recombinante sous le contrôle du promoteur de type T7 ; et un acide nucléique qui code pour une ARN polymérase de type T7 possédant un acide aminé pourvu d'une identité de séquence de 40 % ou plus par rapport à une séquence d'acides aminés représentée par la séquence SEQ ID NO:9.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5385834A (en) * 1993-08-13 1995-01-31 Georgia Tech Research Corporation Mutant T7 RNA polymerase GP1(lys222) exhibiting altered promoter recognition
US5869320A (en) * 1984-03-30 1999-02-09 Brookhaven Science Associates Llc Cloning and expression of the gene for bacteriophage T7 RNA polymerase
JP2002532095A (ja) * 1998-12-11 2002-10-02 アクゾ・ノベル・エヌ・ベー 安定性を増大させたｒｎａポリメラーゼ変異体
WO2014013067A1 (fr) * 2012-07-19 2014-01-23 RUHR-UNIVERSITäT BOCHUM Variants de promoteur t7 et leurs procédés d'utilisation
WO2016049492A1 (fr) * 2014-09-26 2016-03-31 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Vecteurs d'expression à base de virus et leur utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5869320A (en) * 1984-03-30 1999-02-09 Brookhaven Science Associates Llc Cloning and expression of the gene for bacteriophage T7 RNA polymerase
US5385834A (en) * 1993-08-13 1995-01-31 Georgia Tech Research Corporation Mutant T7 RNA polymerase GP1(lys222) exhibiting altered promoter recognition
JP2002532095A (ja) * 1998-12-11 2002-10-02 アクゾ・ノベル・エヌ・ベー 安定性を増大させたｒｎａポリメラーゼ変異体
WO2014013067A1 (fr) * 2012-07-19 2014-01-23 RUHR-UNIVERSITäT BOCHUM Variants de promoteur t7 et leurs procédés d'utilisation
WO2016049492A1 (fr) * 2014-09-26 2016-03-31 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Vecteurs d'expression à base de virus et leur utilisation

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHEN, Z. ET AL.: "Information theory based T7-like promoter models: classification of bacteriophages and differential evolution of promoters and their polymerases", NUCLEIC ACIDS RES., vol. 33, no. 19, 2005, pages 6172 - 6187, XP002475065, doi:10.1093/nar/gki915 *
EVANS, PD ET AL.: "LITMUS: multipurpose cloning vectors with a novel system for bidirectional in vitro transcription", BIOTECHNIQUES, vol. 19, no. 1, 1995, pages 130 - 135 *
IMBURGIO, D. ET AL.: "Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants", BIOCHEMISTRY, vol. 39, no. 34, 2000, pages 10419 - 10430, XP055125483, doi:10.1021/bi000365w *
KLEMENT, JF ET AL.: "Discrimination between bacteriophage T3 and T7 promoters by the T3 and T7 RNA polymerases depends primarily upon a three base-pair region located 10 to 12 base-pairs upstream from the start site", J. MOL. BIOL., vol. 215, no. 1, 1990, pages 21 - 29, XP009047151, doi:10.1016/S0022-2836(05)80091-9 *
LEE, SS ET AL.: "Two base pairs at -9 and -8 distinguish between the bacteriophage T7 and SP6 promoters", J. BIOL. CHEM., vol. 268, 1993, pages 19299 - 19304, XP055617287 *
LIEBER, A. ET AL.: "Stable high-level gene expression in mammalian cells by T7 phage RNA polymerase", METHODS IN ENZYMOLOGY, vol. 217, 1993, pages 47 - 66 *
LIN, YR ET AL.: "Characterization of a new phage, termed phi 318, which is specific for Vibrio alginolyticus", ARCH. VIROL., vol. 157, no. 5, 2012, pages 917 - 926, XP035048347 *
LIU, W. ET AL.: "Genome sequences characterizing five mutations in RNA polymerase and major capsid of phages phi A318 and phi As51 of Vibrio alginolyticus with different burst efficiencies", BMC GENOMICS, vol. 15, 505, 2014, pages 1 - 12, XP021189957 *

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