JP2021040485A - Expression systems of recombinant proteins, expression vectors, recombinant cells and methods for producing recombinant proteins - Google Patents

Abstract

To provide expression systems with higher recombinant protein expression efficiency than the conventional expression systems.SOLUTION: Disclosed is an expression system for a recombinant protein, which comprises: a T7-like promoter comprising a promoter sequence having a sequence identity of not less than 50% and not more than 95% to a T7 promoter sequence represented by SEQ ID NO:8; a nucleic acid encoding the recombinant protein under the control of the T7-like promoter; and a nucleic acid encoding a T7-like RNA polymerase comprising an amino acid sequence having a sequence identity of 40% or more to the amino acid sequence represented by SEQ ID NO:9.SELECTED DRAWING: None

Description

The present invention relates to a recombinant protein expression system, an expression vector, recombinant cells, and a method for producing a recombinant protein. More specifically, the present invention relates to an expression system or the like in which a novel T7-like promoter and T7-like RNA polymerase are combined.

Expression systems for recombinant proteins hosting mammals, eukaryotes and prokaryotes have been established to some extent and are also used in industrial production. In particular, a recombinant protein expression system (T7 recombinant protein expression system) that combines a T7 promoter and T7 RNA polymerase is used as an expression system capable of expressing and obtaining a large amount of recombinant protein, and the T7 system and pET It can also be obtained from Novagen as a system.

As an improved technique for the T7 recombinant protein expression system, Patent Document 1 describes the expression of a promoter-driven protein derived from T7 having an operator sequence downstream of the T7 promoter sequence and a further operator sequence upstream of the T7 promoter sequence. The system is disclosed.

Further, as an expression system other than the T7 recombinant protein expression system, Patent Document 2 describes a protein expression system, which comprises a) T7A1, T7A2, T7A3, λpL, λpR, lacUV5, trp, trc, foA and rrnB. An expression system is disclosed that comprises a promoter selected from the group; and b) a complete parindrome operator sequence.

Special Table 2001-511352 Gazette Japanese Unexamined Patent Publication No. 2017-136072

An object of the present invention is to provide an expression system in which the expression efficiency of a recombinant protein is higher than that of a conventional expression system.

The present inventor uses a T7-like promoter in which the base sequence of the T7 promoter is modified and a T7-like RNA polymerase having a promoter recognition format different from that of the T7 RNA polymerase in combination with a conventional T7 recombinant protein expression system. In 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].
[1]
Recombinant protein expression system
A T7-like promoter containing 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.
Nucleic acids encoding recombinant proteins under the control of the T7-like promoter,
An expression system comprising a nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence having an amino acid sequence of 40% or more with the amino acid sequence shown in SEQ ID NO: 9.
[2]
The expression system according to [1], wherein the above promoter sequence is a thymine (T) residue in which 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.
[3]
The transcription efficiency of the T7-like promoter when combined with T7-like RNA polymerase having the amino acid sequence shown by SEQ ID NO: 9 is T7 having the T7 promoter sequence shown by SEQ ID NO: 8 and the amino acid sequence shown by SEQ ID NO: 9. The expression system according to [1] or [2], which is at least twice or more the transcription efficiency when combined with such RNA polymerase.
[4]
The expression system according to any one of [1] to [3], wherein the T7-like RNA polymerase has an activity of performing transcription by a T7-like promoter containing the nucleotide sequence shown in SEQ ID NO: 1.
[5]
A T7-like promoter containing 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.
An expression vector comprising 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 an amino acid sequence having 40% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9.
[7]
The expression vector according to [5] or [6], which is a plasmid vector or a viral vector.
[8]
A reaction mixture for recombinant cell or cell-free protein synthesis, which comprises the expression system according to any one of [1] to [4].
[9]
Recombinant cells transformed with the expression vector according to any one of [5] to [7].
[10]
The recombinant cell according to [8] or [9], which is a prokaryote.
[11]
The prokaryotes are microorganisms belonging to a genus selected from the group consisting of Escherichia, Vibrio, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. The recombinant cell according to [10].
[12]
The recombinant cell according to [8] or [9], which is a eukaryote.
[13]
The recombinant cell according to [12], wherein the eukaryote is a yeast, filamentous fungus or insect cell.
[14]
The yeast belongs to a genus selected from the group consisting of the genus Saccharomyces, the genus Schizosaccharomyces, the genus Cryberomyces, the genus Tricosporone, the genus Ciwaniomyces, the genus Pikia, the genus Candida, the genus Yarowia and the genus Hanzenula, [13]. Recombinant cells according to.
[15]
The method for producing a recombinant protein using the recombinant cell according to any one of [8] to [14].

According to the present invention, it is possible to provide an expression system having a higher expression efficiency of a recombinant protein than a conventional expression system.

It is a schematic diagram which shows the base sequence of the T7 promoter (pT7, SEQ ID NO: 8) and the T7-like promoter (pT7L1, SEQ ID NO: 1). It is a schematic diagram which shows the result of multiple alignment of the amino acid sequences of T7 RNA polymerase (T7RNAP, SEQ ID NO: 10) and T7-like RNA polymerase (T7LRNAP, SEQ ID NO: 9). It is a schematic diagram which shows the fragment containing the T7 promoter synthesized in the Example. It is a schematic diagram which shows the vector map of the expression vector p15A-HGFP-pT7 / T7RNAP. It is a schematic diagram which shows the fragment containing the T7-like promoter (pT7L1) synthesized in the Example.

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

[Expression system]
The expression system according to the present embodiment is a system for expressing a target recombinant protein, and is a T7-like promoter, a nucleic acid encoding a recombinant protein under the control of the T7-like promoter, and a T7-like RNA polymerase. Includes nucleic acids encoding.

[T7-like promoter]
In the present specification, the T7-like promoter refers to a promoter sequence having a sequence identity of 50% or more and 95% or less with the T7 promoter sequence represented by SEQ ID NO: 8 (hereinafter, also referred to as “consensus region corresponding sequence”). It means a base sequence having promoter activity including. The T7-like promoter is a promoter that is recognized by T7-like RNA polymerase having the amino acid sequence shown by SEQ ID NO: 9 and can be involved in transcription initiation of the gene sequence downstream thereof, while the amino acid sequence shown by SEQ ID NO: 10 For T7 RNA polymerase having, it is a promoter that is not involved in or hardly participates in transcription initiation.

The T7-like promoter according to the present embodiment has a transcription efficiency when combined with a T7-like RNA polymerase having the amino acid sequence represented by SEQ ID NO: 9, which is the T7 promoter sequence represented by SEQ ID NO: 8 and the amino acid represented by SEQ ID NO: 9. Compared with the transcription efficiency when combined with T7-like RNA polymerase having a sequence, it is preferably at least 2 times or more, more preferably 10 times or more, 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 any one containing a sequence corresponding to the consensus region, and those further containing a base sequence affecting the promoter activity before and after (upstream and / or downstream) the sequence are not excluded.

The sequence corresponding to the consensus region has a sequence identity of 50% or more and 95% or less with the T7 promoter sequence shown by SEQ ID NO: 8. The T7 promoter sequence represented by SEQ ID NO: 8 is a consensus region of the T7 promoter utilized in the conventional T7 recombinant protein expression system. Therefore, the consensus region equivalent sequence is modified by substituting, deleting, inserting and / or adding one or more residues to the consensus region of the conventional T7 promoter (T7 promoter sequence shown by SEQ ID NO: 8). It can also be said that it is a promoter.

In the present specification, the sequence identity means the ratio (%) of the residues that match when the base sequence or amino acid sequence to be compared is multiple-aligned. In multiple alignment, in order to make the base sequence or amino acid sequence comparable to each other, the base sequence is aligned by appropriately inserting a blank (gap) so that the corresponding base sequence or amino acid sequence portion is lined up. Say something. A known multiple alignment creation program can be used for multiple alignment. For example, Clustal W, Clustal X, BLAST programs and the like are preferably available.

This consensus region of the T7 promoter is important because it affects the efficiency of promoter recognition and transcription by RNA polymerase, but the region other than the consensus region does not significantly affect the efficiency of promoter recognition and transcription, and therefore is other than the consensus region. The base sequence of the region does not have to be the same as the base sequence of the T7 promoter. The sequence may be modified according to the host in a timely manner according to the host used for transformation.

In the consensus region equivalent sequence, it is preferable that the residue corresponding to the 9th cytosine (C) residue of the T7 promoter sequence shown in SEQ ID NO: 8 is a thymine (T) residue. This increases the expression efficiency of the recombinant protein when combined with T7-like RNA polymerase. The consensus region equivalent sequence further states that the residue corresponding to the 6th thymine (C) residue of the T7 promoter sequence shown in SEQ ID NO: 8 is an adenine (A) residue, and that the 8th adenine (A) residue. ) The residue corresponding to the base is a thymine (T) residue, the residue corresponding to the 10th thymine (T) residue is a guanine (G) residue, and the 19th guanine ( The residue corresponding to the G) residue is an adenine (A) residue, the residue corresponding to the 20th guanine (G) residue is a thymine (T) residue, and the 21st. It is more preferable that at least one of the residues corresponding to the guanine (G) residue of the above is an adenine (A) residue, and it is further preferable that all of them are satisfied.

As a specific example when the sequence corresponding to the consensus region is a sequence having all of the above modifications, for example, a T7-like promoter (pT7L1) having the nucleotide sequence shown in SEQ ID NO: 1 can be mentioned. As a specific example of the case where the sequence corresponding to the consensus region is a sequence having a part of the above modification, for example, a T7-like promoter (pT7L2 to pT7L7) having the nucleotide sequence shown in any of SEQ ID NOs: 2 to 7 can be mentioned. Can be done.

The sequence identity between the T7-like promoter (pT7L1) having the nucleotide sequence shown in SEQ ID NO: 1 and the T7 promoter having the nucleotide sequence shown in SEQ ID NO: 8 is 52% (11/21) (see FIG. 1). ..

In the T7-like promoter according to the present embodiment, the sequence identity corresponding to the consensus region with the T7 promoter sequence represented by SEQ ID NO: 8 may be 50% or more and 95% or less, and 50% or more and 90% or less. It may be 50% or more and 85% or less, and may be 55% or more and 80% or less.

Further, in the T7-like promoter according to the present embodiment, the sequence identity corresponding to the consensus region with the T7-like promoter (pT7L1) represented by SEQ ID NO: 1 was 60% or more, often 65% or more. It may be 70% or more, 80% or more, 85% or more, or 90% or more.

[T7-like RNA polymerase (T7LRNAP)]
In the present specification, T7-like RNA polymerase (hereinafter, also referred to as “T7LRNAP”) is an RNA polymerase having an amino acid sequence having an amino acid sequence of 40% or more with the amino acid sequence shown by SEQ ID NO: 9. Means. The T7-like RNA polymerase is an RNA polymerase having an activity of performing transcription by the T7-like promoter shown in SEQ ID NO: 1. Specific examples of the T7-like RNA polymerase include RNA polymerase having the amino acid sequence shown in SEQ ID NO: 9.

In FIG. 2, the amino acid sequences of T7 RNA polymerase (T7RNAP, SEQ ID NO: 10) used in the conventional T7 recombinant protein expression system and T7-like RNA polymerase (T7LRNAP, SEQ ID NO: 9) according to one embodiment are multi-aligned. It is a schematic diagram which shows the result. The sequence identity of T7RNAP and T7-like RNAP was 33% and the similarity was 48%.

As the T7-like RNA polymerase according to the present embodiment, the sequence identity with the amino acid sequence shown in SEQ ID NO: 9 is preferably 60% or more, more preferably 70% or more, and 80% or more. It is even more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[Recombinant protein]
The recombinant protein produced by the expression system according to the present embodiment (hereinafter, may also be referred to as “target protein”) may be any protein as long as it is intended to be obtained, but on an industrial scale. It is preferable that the product is intended for production in Japan. For example, proteins that can be used for industrial purposes, proteins that can be used for medical purposes, structural proteins, and the like can be mentioned. Specific examples of proteins that can be used for industrial or medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used for vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, etc. Examples include allergens, full-length antibodies or antibody fragments or derivatives. Specific examples of structural proteins include spider silk, silk moth silk, keratin, collagen, elastin and resilin, and proteins derived from these.

As a protein derived from the fibroin-like protein spider silk or silkworm silk, for example, _{a protein containing a domain sequence represented by the formula 1: [(A) n} motif-REP] _m (here, (A) in the formula 1). _{The n} motif shows an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues is 80% or more of the total number of amino acid residues in the _{n motif. REP is 10 to 200 amino acid residues.} Indicates an amino acid sequence composed of groups. M indicates an integer of 8 to 300. Multiple existing (A) _n motifs may have the same amino acid sequence or different amino acid sequences. Multiple REPs may be present. The amino acid sequences may be the same as each other, or different amino acid sequences may be used.) Specifically, a protein containing the amino acid sequence shown by SEQ ID NO: 11 (PRT410) can be mentioned.

As a collagen-derived protein, for example, a protein _{containing a domain sequence represented by the formula 2: [REP2] o} (here, in the formula 2, o represents an integer of 5 to 300. REP2 is Gly-X-Y. Indicates an amino acid sequence composed of, and X and Y indicate arbitrary amino acid residues other than Gly. Multiple REP2s may have the same amino acid sequence or different amino acid sequences.) .. Specifically, a protein containing the amino acid sequence shown in 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 human collagen type 4 partial sequence (NCBl Genebank accession number: CAA5635.1, GI: 3702452) obtained from the NCBI database. The amino acid sequence (tag sequence and hinge sequence) shown by SEQ ID NO: 100 is added to the N-terminal of the amino acid sequence from the 301st residue to the 540th residue.

As a protein derived from resilin, for example, a protein _{containing a domain sequence represented by the formula 3: [REP3] p} (where, in formula 3, p represents an integer of 4 to 300. REP3 is Ser-J-J-1. The amino acid sequence composed of Tyr-Gly-U-Pro is shown. J indicates an arbitrary amino acid residue, and it is particularly preferable that it is an amino acid residue selected from the group consisting of Asp, Ser and Thr. U is arbitrary. It is preferable that it is an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser. The plurality of REP3s may have the same amino acid sequence or different amino acid sequences. ) Can be mentioned. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 101 can be mentioned. The amino acid sequence shown by SEQ ID NO: 101 replaces Thr at residue 87 with Ser and Asn at residue 95 in the amino acid sequence of lecillin (NCBl Genebank accession number NP 61157, Gl: 24654243). The amino acid sequence (tag sequence and hinge sequence) shown by 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 is replaced with Asp.

Examples of the elastin-derived protein include proteins having an amino acid sequence such as NCBl Genebank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 102 can be mentioned. The amino acid sequence shown by SEQ ID NO: 102 is the amino acid sequence shown by SEQ ID NO: 100 at the N-terminal of the amino acid sequence from the 121st residue to the 390th residue of the amino acid sequence of accession number AAC98395 of Genebank of NCBl (tag sequence). And hinge arrangement) is added.

Examples of the keratin-derived protein include Type I keratin of Capra hilcus. Specifically, a protein containing the amino acid sequence shown by SEQ ID NO: 103 (amino acid sequence of NCBl Genebank accession number ACY30466) can be mentioned.

The expression system according to the present embodiment includes at least (i) a nucleic acid encoding a T7-like promoter, (ii) a nucleic acid encoding a recombinant protein under the control of the T7-like promoter, and (iii) a nucleic acid encoding a T7-like RNA polymerase. ,, And the specific embodiment is not particularly limited. For example, all of (i) to (iii) may be integrated into the host's genome, at least one of (i) to (iii) is integrated into the host's genome, and the others are extrachromosomal DNA (eg, iii). It may be retained in the host as a plasmid), or all of (i) to (iii) may be retained in the host as extrachromosomal DNA (for example, a plasmid). Further, the fact that the nucleic acid encoding the recombinant protein is under the control of the T7-like promoter means that the nucleic acid is transcribed by the function of the T7-like promoter.

[Expression vector]
The expression system according to this embodiment preferably functions as an expression vector. That is, the expression vector according to the present embodiment includes (i) a T7-like promoter containing a promoter sequence having a sequence identity of 50% or more and 95% or less with the T7 promoter sequence represented by SEQ ID NO: 8, and (ii) T7. It contains at least a nucleic acid encoding a recombinant protein under the control of a promoter. The expression vector according to the present embodiment further contains (iii) a nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence having an amino acid sequence of 40% or more with the amino acid sequence shown in SEQ ID NO: 9. May be good.

When the expression vector according to the present embodiment does not contain a nucleic acid encoding T7-like RNA polymerase having an amino acid sequence having an amino acid sequence having 40% or more sequence identity with the amino acid sequence shown in (iii) SEQ ID NO: 9. For example, it can be used as an expression system according to the present invention by introducing a nucleic acid (iii) as a genomic or extrachromosomal DNA in advance and combining it with a host capable of expressing T7-like RNA polymerase.

The expression vector according to this embodiment may further contain (i) one or more regulatory sequences operably linked to a T7-like promoter. Specific examples of regulatory sequences include sequences that control the expression of recombinant proteins in a host (eg, enhancer, ribosome binding sequence, terminator (transcription termination sequence), etc.), and are appropriately used according to the type of host. You can choose.

As the expression vector, any vector can be used as long as it can be autonomously replicated in the host cell or can be integrated into the genomic DNA of the host cell. For example, a plasmid vector, a virus vector, a cosmid vector, or a phosmid vector can be used. , And an artificial chromosomal vector, etc., can be appropriately selected depending on the host cell.

Expression vectors include, for example, pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (). QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. 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 [Escherichia] Prepared from 5408)], prepared from pGHA2 [Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), JP-A-60-221091 No.], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. , 172, 2392 (1990)], pGEX (manufactured by Virus), pACYC184 (manufactured by Nippon Gene Co., Ltd., product number 313-02201), pET system (manufactured by Novagen), YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 ( ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, pPICZ B, the virus that infects night-stealing baculoviridae insects: Examples include baculoviruses such as nuclear polyhedrosis virus (Baculovirus Expression Virus, A Laboratory Manual, WH Freeman and Company, New York (1992)) and the like.

The expression vector according to the present invention preferably further contains a selectable marker gene for selection of transformed recombinant cells. For example, in prokaryotes, resistance genes to various drugs such as tetracycline, ampicillin, and kanamycin can be used as selectable marker genes. Recessive selectable markers that can complement gene mutations involved in auxotrophy can also be used. In eukaryotes such as yeast, a resistance gene to geneticin can be used as a selectable marker gene, and a gene that complements a gene mutation involved in auxotrophy, a selectable marker such as LEU2, URA3, TRP1, or HIS3 is also used. it can. In filamentous fungi, as selectable marker genes, niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6,386-389, (1984)), sC (Gene, Gene, 84,329-334, (1989)), ptrA (BiosciBiotechnol Biochem, 64,1416-1421, (2000)), pyrG (BiochemBiophyss Res Commun, 112,284-289, (1983)), amdS (Gene, Gene) 205-221, (1983)), aureobasidin resistance gene (Mol Gen Genet, 261,290-296, (1999)), benomil resistance gene (Proc Natl Acad Sci USA, 83, 4869-4873 (1986)) A marker gene selected from the group consisting of the hyglomycin resistance gene (Gene, 57, 21-26, (1987)), a leucine-requiring complementary gene, and the like can be mentioned. When the host is an auxotrophic mutant, a wild-type gene that complements the auxotrophy can be used as a selectable marker gene.

[Recombinant cells]
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 the host with the expression vector according to the present embodiment.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

Examples of hosts for prokaryotes such as bacteria include microorganisms belonging to the genus Escherichia, Vibrio, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, etc. Can be done.

Examples of microorganisms belonging to the genus Escherichia include Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BL21 (Merck Millipore), and 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 K5 (ATCC 23506), Escherichia Coli KY3276, Escherichia Coli G. ), Escherichia coli No. 49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Tuner (Novagen), Escherichia coli Tuner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W1485, Escherichia coli ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue and the like can be mentioned.

Non-pathogenic microorganisms belonging to the genus Vibrio are desirable, for example, Vibrio avalonicus ATCC27390, V. apatatus ATCC19263, Vibrio aerogenes ATCC700797, Vibrio aerogenes. V. aestuarianus ATCC35048, Vibrio arginolytics ATCC14582, Vibrio argosus ATCC14390, Vibrio angiralum (V. angilarum) Vibrio angiralum (V. angilarum) BAA-606, Vibrio Cambellii ATCC25920, Vibrio carchariae ATCC35084, Vibrio colorilytics ATCC BAA-450, Vibrio 43 Vibrio・ V. cyclitropicus ATCC700982, Vibrio cyclocites ATCC14635, Vibrio diazotropicus ATCC33466, Vibrio fibrio ge, vibrio fisci , Vibrio Harveyoli ATCC700680, Vibrio Harveyi ATCC14126, Vibrio Hispanica ATCC51589, Vibrio Ichitio Enteri (V. V. iliopiscarius ATCC51760, V. lentus ATCC BAA-539, V. liquefaciens ATCC1 7058, Vibrio Rogain (V. logei ATCC15382, Vibrio Marinagiris ATCC14398, Vibrio Marinofulbus ATCC14395, Vibrio Marinobulgaris (V. marinovulgaris), Vibrio Marinobulgaris (V. marinovulgaris) ATCC14. V. methschnikovii ATCC7708, V. mytili ATCC51288, V. natriegens NBRC15636 (ATCC14048), Vibrio Navalensis (ATCC14048), Vibrio Navalensis (ATCC14048), Vibrio Navalensis (V.・ V. nigripulchritudo ATCC27043, Vibrio ordalii ATCC33509, Vibrio orientalis ATCC33933, Vibrio pectenicitida (V. vibrio pectenitide) ATCC33504, Vibrio penaechida (V. penaeicida) ATCC51841, Vibrio pontics (V. pontis) ATCC14391, Vibrio proteolytics (V. V. salmonicida ATCC43839, Vibrio siroii ATCC BAA-91, Vibrio splendidas ATCC33125, Vibrio tyrosinatics (V. tyrosinatics) ATCC33125, Vibrio tyrosinatics (V. tyrosinatics) viscos) ATCC BAA-105, Vibrio wodanis ATCC BAA-104, etc. Can be done.

Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, Brevibacillus bolsterensis, Brevibacillus centroporus Brevibacillus formosas, Brevibacillus invocatus, Brevibacillus lachilosporus, Brevibacillus limnofilus, Brevibacillus parabis. , Brevibacillus Reus Zeri, Brevibacillus Thermolver, Brevibacillus Brevis 47 (FERM BP-1223), Brevibacillus Brevis 47K (FERM BP-2308), Brevibacillus Brevis 47-5 (FERM BP-1664), Brevi Brevibacillus chocinensis 47-5Q (JCM8975), Brevibacillus chocinensis HPD31 (FERM BP-1087), Brevibacillus chocinensis HPD31-S (FERM BP-6623), Brevibacillus chocinensis HPD31-OK (FERM BP-) 4573), Brevibacillus chocinensis SP3 strain (manufactured by Takara), and the like.

Examples of microorganisms belonging to the genus Serratia include, for example, Serratia liquiefacience ATCC14460, Serratia entomophila, Serratia ficiaria, Serratia ficiaria, Serratia ficaria, Serratia ficaria. (Serratia grimesii), Serratia proteamaculans, Serratia odorifera, Serratia primushika (Serratia primusica), Serratia primusica, etc.

Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens.

Examples of microorganisms belonging to the genus Microbacterium include Microbacterium / Ammonia Philum ATCC15354 and the like.

Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum (Corinebacterium glutamicum) ATCC14020, Brevibacterium flavum (Corinebacterium glutamicum ATCC14067) ATCC13826, ATCC14067, Brevibacterium immariophyllum. (Brevibacterium immunophilium) ATCC14068, Brevibacterium lactofermentum (Colinebacterium glutamicum ATCC13869) ATCC13665, ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium saccharolitamic, Brevibacterium Thiogenitalis ATCC19240, Brevibacterium album ATCC15111, Brevibacterium serinum ATCC15112 and the like can be mentioned.

Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes ATCC6781, ATCC6782, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC140. Lamb (Corynebacterium acetoacidophilum) ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolitamicum ATCC21511, Corynebacterium carnae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC130 Examples thereof include Corynebacterium melanocera ATCC17965, Corynebacterium thermoaminogenes AJ12340 (FERMBP-1539), Corynebacterium herculis ATCC13868 and the like.

Pseudomonas microorganisms belonging to the genus Pseudomonas include, for example, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas esudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas (Pseudomonas sp.) D-0110 and the like can be mentioned.

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

Examples of yeast include the genus Saccharomyces, the genus Schizoccharomyces, the genus Kluyveromyces, the genus Trichosporon, the genus Trichosporon, the genus Candida, the genus Candida, and the genus Candida. , Yeasts belonging to the genus Pichia, the genus Pichia, and the like. More specifically, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces marxianus (Kluyveromyces marxianus), Trichosporon pullulans (Trichosporon pullulans), Shiwaniomaisesu - Albius (Schwanniomyces alluvius), Swanniomyces occidentalis, Candida utilis, Pichia pastris (Pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia pichia (Pichia polymorpha), Pichia stypitis, Yarrowia lipolytica, Hansenula polymorpha, and the like.

Filamentous fungi include, for example, the genus Acremonium, the genus Aspergillus, the genus Ustirago, the genus Trichoderma, the genus Neurospora, the genus Fusalium, the genus Fusaria, and the genus Fusarium. Penicillium, Myceliophtra, Botryts, Magnaporthe, Mucor, Metalithium, Monascus, Monascus , And bacteria belonging to the genus Risomcoa.

Specific examples of filamentous fungi include Aspergillus aspergillus, Aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus aspergillus Aspergillus oryzae, Aspergillus sake, Aspergillus sojae, Aspergillus tubignis, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus Parashichikusu (Aspergillus parasiticus), Aspergillus Fikumu (Fikyuumu) (Aspergillus ficuum), Aspergillus Fenikusu (Aspergillus phoeicus), Aspergillus Foechizusu (Fechidasu) (Aspergillus foetidus), Aspergillus Furabusu (Aspergillus flavus), Aspergillus fumigatus (Aspergillus fumigatus ), Aspergillus japonics, Aspergillus japonicus, Trichoderma viride, Trichoderma harzianum, Trichoderma harzianum, Trichoderma risseim Aspergillus, Aspergillus, Aspergillus cellulophyllum, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus, Aspergillus. ora crassa), fusalium oxysporus, fusalium graminearum, fusalium venenatum, fumicola insolence penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium penicillium (Penicillium camemberti), Penicillium Kanesensu (Penicillium canescens), Penicillium Emerusoni (Penicillium emersonii), Penicillium funiculosum (Penicillium funiculosum), Penicillium Gurizeorozeumu (Penicillium griseoroseum), Penicillium Papurogenamu (Penicillium purpurogenum), Penicillium Rokeforuchi (Penicillium roqueforti), Myceriophtaora thermofilm, Mucor ambigus, Mucor cyllineroides, Mucor cyrcinelloides, Mucor cyrcinelloides, Mucoa filalis・ Inaequisporus, Mucor inaequisporus, Mucor oblongillilipticus, Mucor racemosus, Mucor recurbus, Mucor recurvus, Mucor recurvus, Mucor recurvus Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium, Penicillium arrhizus) and the like.

Examples of the insect cell include a winged insect cell, more specifically, an insect cell derived from Prodenia frugiperda such as Sf9 and Sf21, and a cabbage looper (Trichoplusa) such as High 5. Examples include insect cells derived from ni).

As a method for introducing an expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, the method of Cohen et al. (Calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], Proporation method [Method described in JP-A-63-248394, Gene, 17, 107 (1982), 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), Takagi et al. (Agric. Biol. Chem. , 1989, 53: 3099-3100), Okamoto et al. (Bioscii. Biotechnol. Biochem., 1997, 61: 202-203), Electroporation method (Methods Enzymol., 194,182 (1990)), Lithium acetate. Method (J. Bacteriol., 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978), Competitive Method [J. et al. Mol. Biol. , 56: 209 (1971)] and the like.

In the recombinant cell according to the present embodiment, the expression system according to the present embodiment or an expression vector containing the expression system may be integrated into a chromosome (chromosomal DNA).

As a method for incorporating the expression system according to the present embodiment or an expression vector containing the expression system into the host chromosome, a known method can be used, and for example, a recombination mechanism in double strand cleavage repair of λ phage is applied. Examples thereof include the λ red method, the Red / ET homologous recombination method, and the transfer method using the transposon activity using pUT-mini Tn5. Further, for example, a “transposon gene transfer kit: pUTmini-Tn5 Kit” manufactured by Biomedal Co., Ltd. can be used and integrated into the host chromosome according to the method described in the kit.

The recombinant cells transformed with the expression vector according to the present invention can be selected by plaque hybridization, colony hybridization or the like using a probe that selectively binds to the nucleic acid contained in the expression vector. As the probe, a probe obtained by modifying a partial DNA fragment amplified by the PCR method with a radioisotope or digoxigenin based on the nucleotide sequence information of the expression vector according to the present invention can be used.

[Method for producing recombinant protein]
By expressing a nucleic acid encoding a protein of interest in a recombinant cell containing the expression system according to the present invention, the protein of interest can be produced. As an expression method, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. When expressed by yeast, animal cells, or insect cells, the desired protein can be obtained as a polypeptide to which a sugar or sugar chain is added.

The target protein is produced, for example, by culturing recombinant cells 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 the protein from the culture medium. be able to. The method for culturing the recombinant cells according to the present invention in a culture medium can be carried out according to a method usually used for culturing a host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for the recombinant cells according to the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the recombinant cells. However, as long as the medium can efficiently culture the recombinant cells, either a natural medium or a synthetic medium may be used.

The carbon source may be any assimilated by the recombinant cells, such as glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolysates, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests can be used.

As the inorganic salt, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

When a marine bacterium is used as a host such as Vibrio, a medium having a high salt concentration is preferable, and for example, a medium containing 0.5 to 5% of NaCl is preferable.

The prokaryotic or eukaryotic culture can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culturing is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during the culture. When culturing recombinant cells transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as needed. For example, when culturing recombinant cells transformed with a vector using the lac promoter for the expression of T7-like RNA polymerase, isopropyl-β-D-thiogalactopyranoside or the like is transformed with the expression vector using the trp promoter. When culturing transformed recombinant cells, indol acrylic acid or the like may be added to the medium.

As the culture medium for insect cells, commonly used TNM-FH medium (manufactured by Harmingen), Sf-900 II SFM medium (manufactured by Life Technologies), ExCell400, ExCell405 (all manufactured by JRH Biosciences), Race' s Insect Medium (Nature, 195, 788 (1962)) and the like can be used.

Insect cells can be cultured under conditions such as a culture medium having a pH of 6 to 7 and a culture temperature of 25 to 30 ° C. for a culture time of 1 to 5 days. In addition, an antibiotic such as gentamicin may be added to the culture medium as needed during the culture.

For example, when the protein of interest is produced in the recombinant cell or on the extracellular membrane of the recombinant cell, 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 Protein., 4, 1288 (1990)), or JP-A-5-336963, International Publication No. 94/23021, etc. By applying it mutatis mutandis, the protein of interest can be changed to be actively secreted outside the recombinant cell. That is, the target protein is actively secreted outside the recombinant cell by expressing the target protein in the form of adding a signal peptide to the polypeptide containing the active site of the target protein using a gene recombination method. be able to.

The protein of interest produced by recombinant cells containing the expression system according to the present invention can be isolated and purified by a method usually used for isolation and purification of proteins. For example, when the target protein is expressed in a lysed state in cells, after the culture is completed, the recombinant cells are collected by centrifugation, suspended in an aqueous buffer solution, and then an ultrasonic crusher or a French press. , Mantongaulin homogenizer, dynomil and the like to disrupt the recombinant cells to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting-out method using sulfuric acid, a desalting method, or an organic solvent Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography method, hydrophobic chromatography method using resins such as butyl Sepharose and phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis Purified preparations can be obtained by using the above methods alone or in combination.

As the above chromatography, column chromatography using phenyl-toyopearl (Tosoh), DEAE-toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

When the target protein is expressed by forming an insoluble substance in the cells, the recombinant cells are similarly recovered, crushed, and centrifuged to insolubilize the target protein as a precipitate fraction. Collect the body. The recovered insoluble form of the target protein can be solubilized with a protein denaturing agent. After the operation, a purified preparation of the desired protein can be obtained by the same isolation and purification method as described above.

When the target protein or a derivative having a sugar chain added to the target protein is secreted extracellularly, the target protein or its derivative can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

[Protein synthesis using reaction mixture for cell-free protein synthesis]
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 the present embodiment is described, for example, in Methods in molecular biology, vol. 267, pp. 169-182, Mol. System. Biol. , 2008, vol. 4, Article No. 220 and Biotechnol. Bioeng. , 2011, vol. 108, pp. The protein of interest can be synthesized by preparing according to the known method described in 1570-1578. For the components of the reaction mixture, refer to, for example, US Pat. Nos. 7,338,789 and 7,351,563, and US Patent Application Publication Nos. 2010/0184135 and 2010/090324. be able to. As the template nucleic acid, DNA encoding the target protein can be used.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.

<1. Construction of expression vector p15A-pT7 / T7RNAP>
(I) Preparation of DNA fragment (1) containing lac UV5 promoter sequence, lac operator sequence and T7 RNA polymerase gene sequence Using the genomic sequence information of BL21 (DE3) (Novagen, product number 69450), the lac UV5 promoter sequence, A 4484 base pair (bp) DNA fragment (1) containing the lac operator sequence and the T7 RNA polymerase (also referred to as "T7RNAP") gene sequence was synthesized and used with the two primers shown in Table 1 (see Table 1). It was amplified by the PCR method. PCR was carried out under the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 30 seconds for 35 cycles.

(Ii) Preparation of DNA fragment (2) containing replication origin sequence and chloramphenicol resistance gene sequence replication origin using pACYC184 (Nippon Gene Co., Ltd., product number 313-02201) having a p15A DNA replication initiation region as a template. The DNA fragment (2) containing the sequence and the chloramphenicol resistance gene sequence was amplified by the PCR method using the two types of primers shown in Table 2 (see Table 2). PCR was carried out under the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 30 seconds for 35 cycles.

(Iii) Preparation of DNA fragment (3) containing pelB signal sequence, T7 promoter sequence, ribosome binding site (RBS) sequence and T7 terminator sequence Using the nucleotide sequence information of pET22b (+) (Novagen, product number 69744). , PelB signal sequence, T7 promoter sequence, ribosome binding site (RBS) sequence and T7 terminator sequence, 395 bp DNA fragment (3) was synthesized and PCR was performed using the two primers shown in Table 3 (see Table 3). Amplified by the method. PCR was carried out under the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 5 seconds for 35 cycles.

The DNA fragments (1) to (3) prepared in the above (i) to (iii) are ligated using an In-Fusion (registered trademark) cloning system (manufactured by Takara Bio Inc.) to prepare a vector (1). did.

The vector (1) was cleaved with restriction enzymes BglII and SacI and linearized, and ligated and linked by the Overlap extension PCR (OE-PCR) method using the four types of primers shown in Table 4 (see Table 4). An expression vector in which a fragment containing the T7 promoter (see FIG. 3) prepared by amplification was ligated using an In-Fusion (registered trademark) cloning system (manufactured by Takara Bio Co., Ltd.) and the pelB signal sequence was removed. p15A-pT7 / T7RNAP was constructed. The nucleotide sequence of the expression vector p15A-pT7 / T7RNAP is shown in SEQ ID NO: 22. The base sequence was confirmed by the Sanger method.

<2. Construction of expression vector p15A-HGFP-pT7 / T7RNAP>
A DNA fragment (4) containing a sequence encoding Holly green fluorescent protein (HGFP-His) with a 6 × His tag added to the C-terminal using T5-HollyGFP (manufactured by Cosmo Bio Co., Ltd.) as a template is shown in Table 5. Amplified by PCR using different primers (see Table 5). PCR was carried out under the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 10 seconds for 35 cycles.

A fragment obtained by cleaving the expression vector p15A-pT7 / T7RNAP with restriction enzymes NdeI and SacI and a DNA fragment (4) are ligated and expressed using an In-Fusion (registered trademark) cloning system (manufactured by Takara Bio Co., Ltd.). 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.

<3. Synthesis of T7-like RNA polymerase (T7LRNAP) gene>
A DNA fragment (5) containing a base sequence encoding T7LRNAP having the amino acid sequence shown in SEQ ID NO: 9 was synthesized by the following method.

66 types of primers shown in Tables 6 and 7 were designed (see Table 6) and synthesized so as to cover the nucleotide sequence encoding T7LRNAP (synthesis was outsourced to Fasmac Co., Ltd.). Each primer is designed so that the sense strand and the antisense strand alternately overlap by about 20 bases.

A DNA fragment containing a base sequence encoding T7LRNAP by ligating all the primers by the method shown below by the Dual assay PCR (DA-PCR) method or the like using the synthesized primers (T7LRNAP- # 1 to # 66). (5) was acquired.

(First stage: DA-PCR)
First, for T7LRNAP- # 1 to # 56, put in one reaction tube for every eight consecutive primers in ascending order, and for T7LRNAP- # 57 to # 66, one reaction for every four consecutive primers in ascending order. It was placed in a tube, and 25 cycles were performed under the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 5 seconds, respectively. Two outermost primers after ligation were added to each reaction tube in a molar excess of 5 times that of the inner primers.

The obtained reaction product (fragment in which the primer added to the reaction tube is specifically linked) was prepared using Wizard (registered trademark) SV Gel and PCR Clean-Up System (manufactured by Promega) according to the attached manual. Purified.

(Second stage: DA-PCR)
The purified reaction products (T7LRNAP- # 1 to # 8 ligated fragments, T7LRNAP- # 9 to # 16 ligated fragments, T7LRNAP- # 17 to # 24 ligated fragments, and T7LRNAP- (Connected fragments of # 25 to # 32) were added to the new reaction tube by 5 μL each, and 1 μL of primer RNAP_F (10 pmol / μL) and primer T7LRNAP- # 32 (10 pmol / μL) shown in Table 8 were added to the new reaction tube, respectively. Max (registered trademark) Max (manufactured by Takara Bio Co., Ltd.) was added so as to have a final volume of 60 μL, and ultrapure water was carried out under the same conditions as in the first stage DA-PCR.

Similarly, purified reaction products (T7LRNAP- # 33- # 40 ligated fragments, T7LRNAP- # 41- # 48 ligated fragments, T7LRNAP- # 49- # 56 ligated fragments, T7LRNAP- 5 μL each of the ligated fragments of # 57 to # 61 and the ligated fragments of T7LRNAP- # 62 to # 66) were added to another new reaction tube, and further added to the primer RNAP_R (10 pmol / μL) shown in Table 8. Primer T7LRNAP- # 33 (10 pmol / μL) was added to 1 μL each, PrimeSTAR® Max (manufactured by Takara Bio Inc.) to 30 μL, and ultrapure water was added to a final volume of 60 μL, and the first step DA-PCR was added. PCR was performed under the same conditions as above.

The obtained reaction product (fragment in which the first-stage reaction product added to the reaction tube was specifically linked) was prepared using Wizard (registered trademark) SV Gel and PCR Clean-Up System (manufactured by Promega). And purified according to the attached manual.

(Third stage)
Two purified reaction products (a linked fragment of T7LRNAP- # 1 to # 32 and a linked fragment of T7LRNAP- # 33 to # 66), and a restriction enzyme HincII cleaved and linearized by BAP. Dephosphorylated pUC118 (manufactured by Takara Bio Inc., product number 3322) was ligated using an In-Fusion® cloning system (manufactured by Takara Bio Inc.).

The obtained ligated fragment (double-stranded DNA fragment containing the base sequence encoding T7LRNAP) was amplified by the PCR method using primers T7LRNAP- # 1 and T7LRNAP- # 66 after confirming the base sequence by the Sanger method. .. PCR was carried out under the conditions of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 40 seconds for 35 cycles. The reaction product obtained by PCR was purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (manufactured by Promega) according to the attached manual, and a DNA fragment containing a base sequence encoding T7LARNP (). 5) was obtained.

<4. Construction of expression vector p15A-HGFP-pT7 / T7LRNAP>
An expression vector p15A-HGFP-pT7 / T7RNAP was constructed by replacing the nucleotide sequence encoding the T7RNAP gene in the expression vector p15A-HGFP-pT7 / T7RNAP with the nucleotide sequence encoding the T7RNAP gene by the following method.

First, the expression vector p15A-HGFP-pT7 / T7RNAP was amplified by the PCR method using the two types of primers shown in Table 9 (see Table 9) and linearized. PCR was carried out under the conditions of 98 ° C. for 10 seconds, 60 ° C. for 5 seconds, and 72 ° C. for 40 seconds for 35 cycles.

Next, a linearized p15A-HGFP-pT7 / T7RNAP amplified fragment and a DNA fragment (5) containing a base sequence encoding T7RNAP were combined using an In-Fusion (registered trademark) cloning system (manufactured by Takara Bio Inc.). The expression vector p15A-HGFP-pT7 / T7LRNAP was constructed by ligation.

<5. Synthesis of T7-like promoter and construction of expression vector>
(Construction of p15A-HGFP-pT7L1 / T7LRNAP)
The four primers shown in Table 10 (see Table 10) were ligated and amplified by the OE-PCR method to synthesize a DNA fragment (6) containing a T7-like promoter (pT7L1) sequence, a lac operator sequence and an RBS sequence. FIG. 5 is a diagram showing the base sequence of the DNA fragment (6).

Next, an expression vector p15A-HGFP-pT7L1 / T7LRNAP was constructed by substituting the T7 promoter sequence in the expression vector p15A-HGFP-pT7 / T7LRNAP with a T7-like promoter (pT7L1) sequence by the following method.

First, the expression vector p15A-HGFP-pT7 / T7LRNAP was cleaved with restriction enzymes BglII and SacI and linearized. Next, the DNA fragment (6) and the linearized p15A-HGFP-pT7 / T7LRNAP fragment were ligated using an 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.

(Construction of p15A-HGFP-pT7L2-pT7L7 / T7LARNP)
Table 11 shows the T7 promoter sequence in the expression vector p15A-HGFP-pT7 / T7LARNP by the same procedure as for the expression vector p15A-HGFP-pT7L1 / T7RLNP except that the base sequence of the primer used was changed. An expression vector p15A-HGFP-pT7L2 to pT7L7 / T7LRNAP was constructed by substituting the T7-like promoter (pT7L2 to pT7L7) sequence. The nucleotide sequence of the obtained expression vector was confirmed by the Sanger method.

<6. Preparation of HGFP-expressing recombinant cells and analysis of HGFP expression level>
Escherichia coli BL21 strain (Merck Millipore) and Vibrio natriegens NBRC15636 strain were transformed with two types of expression vectors, p15A-HGFP-pT7 / T7RNAP and p15A-HGFP-pT7L1 / T7RNAP, respectively, prepared above. HGFP-expressing recombinant cells were prepared.

The prepared recombinant cells were cultured by the following method, and the expression level of HGFP was analyzed.

HGFP-expressing recombinant cells transformed with Escherichia coli BL21 strain were cultured with shaking at 37 ° C. using 2 mL of LB medium containing 25 μg / mL chloramphenicol.

Vibrio natriegens In HGFP expressing recombinant cells transformed with NBRC15636 strain, 12.5 [mu] g / mL of chloramphenicol NaCl of chloramphenicol and 11.9g / L, 4.7g / L MgCl 2 · 6H 2 O and 0. Using 2 mL of LB medium containing 31 g / L of KCl, the cells were cultured with shaking at 37 ° C.

In each case, when OD ₆₀₀ reached about 0.5, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further shake-cultured at 37 ° C. for 3 hours. After culturing, the cultured cells were collected, and the fluorescence intensity of HGFP near the fluorescence wavelength of 515 nm was measured using a Spectral cell analyzer SA3800 (manufactured by Sony Corporation). The number of cells was measured and the average fluorescence intensity per ^{5 × 10 4 cells was calculated.} The expression level of each HGFP-expressing recombinant cell when the expression level (average fluorescence intensity) of the HGFP-expressing recombinant cell transformed with the BL21 strain with p15A-HGFP-pT7 / T7RNAP, which is a conventional expression system, is 100. The relative values of (average fluorescence intensity) are shown in Table 12.

Compared with the expression vector p15A-HGFP-pT7 / T7RNAP composed of T7 promoter (pT7) and T7 RNA polymerase (T7RNAP), which corresponds to the conventional expression system, the expression system of the present invention, T7-like promoter (pT7L1). ) And the expression vector p15A-HGFP-pT7L1 / T7LRNAP composed of T7-like RNA polymerase (T7RNARP) were confirmed to have a significantly increased expression level in all recombinant cells. In particular, the Escherichia coli BL21 strain increased about 20 times, and the Vibrio naturegens NBRC15636 strain also increased about 1.9 times. It was confirmed that the expression system of the present invention can be applied beyond the genus of the host.

Similar to the above, the expression vector p15A-HGFP-pT7 / T7RNAP and the expression vector p15A-HGFP-pT7L1 to pT7L7 / T7RNAP having 7 types of T7-like promoters are used in a total of 7 types of expression vectors. Company-made product No. 9052) was transformed and cultured in the same manner, and the expression level of HGFP was compared. Each HGFP-expressing recombinant cell when the expression level (average fluorescence intensity) of the HGFP-expressing recombinant cell transformed with the JM109 strain with the expression vector p15A-HGFP-pT7 / T7RNAP, which corresponds to the conventional expression system, is 100. Table 13 shows the relative values of the expression level (average fluorescence intensity) of.

It was confirmed that the expression level of T7-like RNA polymerase in which the 9th C residue of the T7 promoter (pT7) was replaced with a T residue increased 3-fold (particularly, comparison between pT7 and pT7L7). In addition to this, it can be confirmed that the expression level is increased about 8 times by further substituting the 6th C residue of pT7 with the A residue and the 8th A residue with the T residue. (In particular, comparison between pT7 and pT7L5). In addition to these, it was confirmed that the expression level was increased about 10-fold by further substituting the 10th T residue of pT7 with the G residue (particularly, comparison between pT7 and pT7L4). From this, it was further confirmed that the expression level was increased about 15-fold by substituting the 19th and 20th GG residues of pT7 with AT residues (particularly, comparison between pT7 and pT7L2). Furthermore, it was confirmed that the expression level was increased about 20 times by substituting the 21st G residue of pT7 with the A residue (particularly, comparison between pT7 and pT7L1).

Similar to the above, Escherichia coli JM109 strain was similarly transformed and cultured with three types of expression vectors, p15A-HGFP-pT7 / T7RNAP, p15A-HGFP-pT7 / T7RNAP and p15A-HGFP-pT7L1 / T7RNAP, and HGFP By comparing the expression levels of Escherichia coli, the compatibility between the promoter and RNA polymerase was confirmed. Each HGFP-expressing recombinant cell when the expression level (average fluorescence intensity) of the HGFP-expressing recombinant cell transformed with the JM109 strain with the expression vector p15A-HGFP-pT7 / T7RNAP, which corresponds to the conventional expression system, is 100. Table 14 shows the relative values of the expression level (average fluorescence intensity) of.

The T7-like promoter in the expression system of the present invention is not suitable for T7 RNA polymerase, and the expression vector p15A-HGFP-pT7L1 / T7RNAP in which they are combined has an extremely low expression level of HGFP of 13 (about 0.1 times). It was a thing. Similarly, the T7 promoter is not suitable for T7-like RNA polymerase, and the expression vector p15A-HGFP-pT7 / T7LRNAP that combines them has an expression level of HGFP of 41 (about 0.4 times), which is the conventional T7 system. It had dropped to less than half of.

<7. Preparation of spider silk protein (SSP) expression recombinant cells and analysis of SSP expression level>
Expression in which the sequence of the HGFP gene in the expression vectors p15A-HGFP-pT7 / T7RNAP and p15A-HGFP-pT7L1 / T7RNAP is replaced with the sequence of the gene encoding the spider silk protein (SSP) having the amino acid sequence shown in SEQ ID NO: 11. The vectors p15A-SSP-pT7 / T7RNAP and p15A-SSP-pT7L1 / T7RNAP were constructed in the same manner as above.

Escherichia coli BL21 strain was transformed with these expression vectors, and the obtained transformant was cultured under the same conditions as above and expression was induced by adding IPTG. However, the expression induction (culture time after addition of IPTG) was set to 6 hours. After induction of expression, 100 μL of the culture solution was collected, centrifuged to remove the supernatant, and then the precipitated cells were suspended in 100 μL of 1 × Laemmlli buffer, and the suspension was heat-treated at 95 ° C. for 5 minutes. Was done. Then, by adding 1 × Laemmlli buffer, the number of cells per volume between the samples was made uniform. Dilution series of each sample (1-fold, 2-fold and 3-fold dilution) were prepared and subjected to SDS-PAGE.

Spider silk protein (SSP) was confirmed by InVision ™ His-Tag In-Gel Staining Kit (Thermo Fisher Scientific, product number LC6033), which specifically detects His tags. The intensity of the band detected at the expected molecular weight was quantified by ImageJ (NIH). The slope of the calibration curve was determined from the band intensity of the dilution series. The correlation coefficient R ² of the calibration curve of each sample, were all 0.99 or more. The relative value of the slope between the samples was taken as the relative value of the expression level between the samples.

The expression vector p15A-SSP-pT7L1 which is the expression system of the present invention when the expression level of the strain transformed from the BL21 strain with the expression vector p15A-SSP-pT7 / T7RNAP, which corresponds to the conventional expression system, is set to 100. Table 15 shows the relative expression levels of the strains transformed with / T7LRNAP from the BL21 strain.

Even when the protein to be expressed is replaced with spider silk protein (SSP), the expression system of the present invention (expression vector p15A-SSP-pT7L1 / T7LRNAP) composed of a T7-like promoter and T7-like RNA polymerase is similar to HGFP. The expression level was about 5 times that of the conventional expression system (expression vector p15A-SSP-pT7 / T7RNAP).

Claims (15)

Recombinant protein expression system
A T7-like promoter containing 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 the T7-like promoter,
An expression system comprising a nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence having an amino acid sequence of 40% or more with the amino acid sequence shown in SEQ ID NO: 9. The expression system according to claim 1, wherein the promoter sequence is a thymine (T) residue in which 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. The transcription efficiency of the T7-like promoter when combined with T7-like RNA polymerase having the amino acid sequence shown by SEQ ID NO: 9 is T7 having the T7 promoter sequence shown by SEQ ID NO: 8 and the amino acid sequence shown by SEQ ID NO: 9. The expression system according to claim 1 or 2, which is at least twice or more the transcription efficiency when combined with such RNA polymerase. The expression system according to any one of claims 1 to 3, wherein the T7-like RNA polymerase has an activity of performing transcription by a T7-like promoter containing the nucleotide sequence shown in SEQ ID NO: 1. A T7-like promoter containing 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.
An expression vector comprising a nucleic acid encoding a recombinant protein under the control of the T7-like promoter. The expression vector according to claim 5, further comprising a nucleic acid encoding a T7-like RNA polymerase having an amino acid sequence having an amino acid sequence having 40% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9. The expression vector according to claim 5 or 6, which is a plasmid vector or a viral vector. A reaction mixture for recombinant cell or cell-free protein synthesis, which comprises the expression system according to any one of claims 1 to 4. Recombinant cells transformed with the expression vector according to any one of claims 5 to 7. The recombinant cell according to claim 8 or 9, which is a prokaryote. The prokaryote is a microorganism belonging to a genus selected from the group consisting of Escherichia, Vibrio, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. The recombinant cell according to claim 10. The recombinant cell according to claim 8 or 9, which is a eukaryote. The recombinant cell according to claim 12, wherein the eukaryote is a yeast, filamentous fungus or insect cell. 13. Recombinant cells according to. The method for producing a recombinant protein using the recombinant cell according to any one of claims 8 to 14.

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