JP2020031550A - Expression systems and recombinant cells - Google Patents

Abstract

To provide novel, inducible protein expression systems.SOLUTION: Disclosed herein is an expression system comprising a first double-stranded DNA fragment comprising a first expression cassette and a second expression cassette, a second double-stranded DNA fragment comprising a third expression cassette, where the first expression cassette comprises a first promoter and a nucleic acid encoding a first repressor protein under the regulation of the first promoter, the second expression cassette comprises a second promoter, one or more of a first operator, and a nucleic acid encoding an RNA polymerase under the regulation of the second promoter, and the third expression cassette comprises a third promoter and a nucleic acid encoding a protein of interest under the regulation of the third promoter.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an expression system for expressing a target protein and a recombinant cell.

  When artificially expressing a large amount of a heterologous protein, the viability of the host may be significantly impaired. Therefore, in order to maximize the protein productivity per culture solution, a so-called “expression induction system” is used in which cells are grown without expressing the target protein and the expression is switched on after reaching a certain number of cells. Is valid.

  Modern protein-related research has been greatly advanced by an expression induction system equipped with a T7 phage-derived RNA polymerase (hereinafter, referred to as “T7 RNA polymerase”) having excellent characteristics in transcription rate and the like. The expression induction system using T7 RNA polymerase utilizes a lacI gene that controls protein production in response to lactose in the environment. That is, by adding lactose or lactose analog isopropyl-β-thiogalactopyranoside (hereinafter IPTG) to the culture solution, the host cell can be switched from the growth phase to the production phase (Non-Patent Document 1, Non-patent document 2).

F. W. Studier, B .; A. Moffatt, Use of bacteriophase T7 RNA polymerase to direct selective high-level expression of cloned genes, J. et al. Mol. Biol. 189 (1986) 113-130. F. W. Studier, A .; H. Rosenberg, J .; J. Dunn, J .; W. Dubendorff, Use of T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol. 185 (1990) 60-89.

  IPTG is a very expensive and difficult-to-produce compound. Therefore, if IPTG can be replaced with another inexpensive substance, there is a great cost advantage in increasing the scale of culture. Therefore, an object of the present invention is to provide a new inducible protein expression system.

  An expression system according to one aspect of the present invention comprises a first double-stranded DNA fragment containing a first expression cassette and a second expression cassette, and a second double-stranded DNA fragment containing a third expression cassette. ,including. The first expression cassette includes a first promoter and a nucleic acid under the control of the first promoter and encoding a first repressor protein. The second expression cassette includes a second promoter, one or more first operators, and a nucleic acid under the control of the second promoter and encoding an RNA polymerase. The third expression cassette includes a third promoter and a nucleic acid under the control of the third promoter and encoding a protein of interest. The third promoter is recognized by the RNA polymerase. The first repressor protein binds to the one or more first operators in the absence of maltose and represses transcription of the nucleic acid encoding the RNA polymerase, but in the presence of maltose, Do not bind to the above first operator.

  The first repressor protein may be a MalR protein.

  The nucleic acid encoding the first repressor protein may have a base sequence represented by SEQ ID NO: 1.

  The one or more first operators may be an operator having a base sequence represented by SEQ ID NO: 2.

  The first expression cassette may include more than one first operator.

  The RNA polymerase may be a T7 RNA polymerase and the third promoter may be a T7 promoter.

  The second double-stranded DNA fragment may further comprise a fourth expression cassette comprising a fourth promoter and a nucleic acid under the control of the fourth promoter and encoding a second repressor protein. The third expression cassette may further include one or more second operators. The second repressor protein binds to the one or more second operators in the absence of maltose and represses transcription of a nucleic acid encoding the protein of interest, but in the presence of maltose, Does not bind to the second operator.

  A recombinant cell according to one aspect of the present invention includes the above protein expression system, and the above RNA polymerase is exogenous.

  The recombinant cell may be E. coli.

  Recombinant cells may not be able to utilize maltose.

  Recombinant cells may include the first double-stranded DNA fragment in chromosomal DNA and the second double-stranded DNA fragment as extrachromosomal DNA.

  The copy number of the extrachromosomal DNA may be 1 to 700.

  The method for producing a target protein according to one aspect of the present invention includes a step of culturing the above-mentioned recombinant cell in the presence of maltose.

  According to the present invention, a novel inducible protein expression system which does not require IPTG for expression induction and a recombinant cell having the expression system are provided.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram which shows the outline | summary of the expression system which concerns on one Embodiment of this invention. It is a schematic diagram which shows the vector map of SSP expression vector. It is a schematic diagram which shows the vector map of SSP expression vector. It is a schematic diagram which shows the vector map of SSP expression vector. FIG. 4 is a schematic diagram showing a vector map of a plasmid containing T7 RNA polymerase, using a CRIM plasmid as a backbone. It is a schematic diagram which shows the vector map of the expression vector of T7 RNA polymerase. It is a migration photograph of SDS-PAGE in a malQ disruption strain. It is an electrophoresis photograph of SDS-PAGE in a strain having malQ.

[1. Expression system]
The expression system according to one embodiment of the present invention is a maltose-inducible protein expression system for expressing a target protein under induction by maltose. FIG. 1 shows an outline of an expression system according to one embodiment of the present invention. The expression system shown in FIG. 1 includes a first double-stranded DNA fragment containing a first expression cassette and a second expression cassette, and a second double-stranded DNA fragment containing a third expression cassette. . The first expression cassette is an expression cassette for expressing a repressor protein, the second expression cassette is an expression cassette for expressing RNA polymerase, and the third expression cassette is for expressing a target protein. Expression cassette for The second double-stranded DNA fragment optionally contains a fourth expression cassette for expressing the repressor protein.

  As used herein, an expression cassette refers to a double-stranded DNA comprising a single promoter and a nucleic acid encoding a protein under the control of the promoter and which is continuously transcribed by an RNA polymerase. . As used herein, a double-stranded DNA fragment refers to a continuous linear or circular double-stranded DNA fragment containing one or more expression cassettes. The double-stranded DNA fragment may be a linear DNA fragment incorporated into the chromosomal DNA of the host cell, or may be a circular DNA fragment introduced into the host cell as extrachromosomal DNA. In the present specification, a nucleic acid means a double-stranded DNA unless otherwise specified, and a nucleic acid encoding a protein may be a gene or cDNA encoding a protein.

  In the present expression system, the RNA polymerase expressed from the second expression cassette recognizes the third promoter contained in the third expression cassette, and thus is under the control of the third promoter and encodes the target protein The nucleic acid is expressed. In the second expression cassette, the expression of the nucleic acid encoding the RNA polymerase under the control of the second promoter is such that, in the absence of maltose, the first repressor protein expressed from the first expression cassette is 1 Inhibited by binding to one or more first operators. On the other hand, in the presence of maltose, the target protein is expressed by RNA polymerase because the first repressor protein does not bind to the one or more first operators. That is, the expression of the target protein is induced by the addition of maltose. Therefore, the target protein can be produced by culturing a recombinant cell having the present expression system in the presence of maltose.

  In order for the expression system according to the present embodiment to function in a host cell, an RNA polymerase expressed from a nucleic acid encoding an RNA polymerase in the second expression cassette must be an RNA polymerase that is exogenous to the host cell (hereinafter referred to as “foreign DNA polymerase”). And the third promoter must be a promoter that is recognized by the exogenous RNA polymerase and not recognized by the endogenous RNA polymerase. If the third promoter is a promoter that is also recognized by endogenous RNA polymerase, the third promoter is recognized by endogenous RNA polymerase, and the nucleic acid encoding the target protein under the control of the third promoter is Is expressed regardless of the presence or absence of maltose.

<First double-stranded DNA fragment>
The first double-stranded DNA fragment contains a first expression cassette for expressing a repressor protein and a second expression cassette for expressing RNA polymerase. The first expression cassette includes a first promoter and a nucleic acid encoding a first repressor protein. The second expression cassette includes a second promoter, one or more first operators, and a nucleic acid encoding an RNA polymerase.

  The first promoter is not limited as long as it functions in a host cell, and examples thereof include a lacI promoter, a lacUV5 promoter, a tac promoter, a λPL promoter, a λPR promoter, a trp promoter, a bioB promoter, a recA promoter, and a str promoter. rpoA promoter, pBRtet promoter, malT promoter, leu promoter, araBAD promoter, ompF promoter, tyrT promoter, ilvM promoter, cspA promoter, pTEF1 promoter, pADH1 promoter, pTPI1 promoter, pHXT7 promoter, pTDH3 promoter, pPGK1 promoter, pVYK1 promoter, CMV promoter ,as well as lpp promoter. As the first promoter, a promoter that functions constantly in a host cell is preferable. When the host cell is Escherichia coli, promoters that function constantly include a lacI promoter, a tac promoter, a trp promoter, and an lpp promoter. From the viewpoint of supplying a repressor protein that controls the second promoter, the first promoter is more preferably a promoter whose expression is not excessively strong and functions constantly. Usually, a promoter of a repressor protein possessed by a microorganism has the above characteristics, and therefore, a promoter that transcribes the repressor protein, such as the lacI promoter, is more preferable.

  The first repressor protein (hereinafter, the repressor protein is also referred to as “repressor”) is a transcriptional regulator in the maltose operon, and examples of the first repressor include MalR protein. The first repressor is bound to one or more first operators described below in the absence of maltose, and dissociates from the first operator in the presence of maltose. Binding of the first repressor to the first operator prevents binding of the RNA polymerase to a second promoter operably linked to the first operator, resulting in transcription of a nucleic acid under the control of the promoter. Is suppressed. The nucleic acid encoding the first repressor may be a Streptococcus pneumoniae malR gene having the nucleotide sequence represented by SEQ ID NO: 1, and the malR gene and the malR gene are at least 85%, preferably at least 90%, more preferably at least 95%. It may be a nucleic acid having a base sequence having at least% sequence identity.

  The nucleic acid encoding the first repressor in the first expression cassette is under the control of the first promoter, and is constituted by an RNA polymerase (hereinafter, referred to as an endogenous RNA polymerase) that is constantly present in a host cell. Is transcribed. As used herein, a nucleic acid under the control of a promoter refers to a nucleic acid that is downstream of the promoter and whose transcription is initiated by binding of an endogenous or exogenous RNA polymerase to the promoter.

  The second promoter is not limited as long as it functions in the host cell, and any of the above-listed promoters may be used as the first promoter. Specific examples of the second promoter include, for example, a lacUV5 promoter.

  An operator is the sequence to which the repressor binds. The first operator is preferably an operator in the maltose operon, and may be, for example, an operator having a base sequence represented by SEQ ID NO: 2. The second expression cassette comprises one or more first operators, preferably two or more operators, more preferably three or more, to facilitate binding to the first operator by the first repressor. Includes two operators.

  The first operator is operably linked to a second promoter. An operator operably linked to a promoter is defined as a position that is such that binding of an endogenous or exogenous RNA polymerase to the promoter is prevented when the repressor binds to the operator. , Means the deployed operator. The first operator may be arranged such that the first base is located, for example, 150 bp upstream to 450 bp downstream from the transcription start point, and, for example, 106 to 119 bp upstream, 1 to 14 bp downstream, Alternatively, they may be arranged such that the first base is located downstream of 394 to 408 bp. Preferably, each operator is separated by a multiple of about 10.5 bp.

  The RNA polymerase can be any RNA polymerase that is exogenous to the desired host cell. For example, when the host cell is Escherichia coli, the exogenous RNA polymerase includes, for example, DNA-dependent RNA polymerase such as T7 RNA polymerase and T7-like RNA polymerase, and bacteriophage N4 virion RNA polymerase. The RNA polymerase is preferably a DNA-dependent RNA polymerase, more preferably a T7 RNA polymerase or a T7-like RNA polymerase, and still more preferably a T7 RNA polymerase.

  T7-like RNA polymerase has a sequence identity of 40% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more with the amino acid sequence represented by SEQ ID NO: 3. Refers to an RNA polymerase having an amino acid sequence. T7-like RNA polymerase specifically recognizes a T7-like promoter described below. Specific examples of T7-like RNA polymerases are T3 RNA polymerase, SP6 RNA polymerase and other T7-like RNA polymerases. Another specific example of the T7-like RNA polymerase is an RNA polymerase having an amino acid sequence represented by SEQ ID NO: 3.

  The nucleic acid encoding the RNA polymerase is under the control of a second promoter and is transcribed by the endogenous RNA polymerase in the presence of maltose. In the absence of maltose, transcription of the nucleic acid encoding RNA polymerase is suppressed because the first repressor is bound to the first operator and the endogenous RNA polymerase cannot bind to the second promoter.

  The first expression cassette and the second expression cassette can further contain a sequence involved in gene expression, such as a terminator and a ribosome binding site, if necessary. The first double-stranded DNA fragment may be an expression cassette for expressing another protein, such as an expression cassette having an antibiotic resistance gene, as long as it does not interfere with the functions of the first expression cassette and the second expression cassette. May be further included.

  In the first double-stranded DNA fragment, the first expression cassette and the second expression cassette may be connected in a direction in which the transcription directions of the respective cassettes are the same or may be connected in opposite directions. May be. That is, the sense strand of the nucleic acid encoding the first repressor and the sense strand of the nucleic acid encoding the exogenous RNA polymerase are on the same strand among the two strands of the first double-stranded DNA fragment. Or on different chains.

<Second double-stranded DNA fragment>
The second double-stranded DNA fragment contains a third expression cassette for expressing the target protein, and optionally contains a fourth expression cassette for expressing the repressor. The third expression cassette can include a third promoter, a nucleic acid encoding the protein of interest, and, optionally, one or more second operators. The fourth expression cassette includes a fourth promoter and a nucleic acid encoding a second repressor. The fourth expression cassette may be identical to the second expression cassette.

  The third promoter is not limited as long as it is recognized by the above-described exogenous RNA polymerase and not recognized by the endogenous RNA polymerase from the viewpoint of making the present expression system function in host cells. For example, when the host cell is Escherichia coli, examples of the third promoter include a T7 promoter and a T7-like promoter. The third promoter is preferably a T7 promoter or a T7-like promoter, more preferably a T7 promoter. When the third promoter is recognized by the exogenous RNA polymerase, the exogenous RNA polymerase binds to the third promoter, and the nucleic acid encoding the protein of interest under the control of the third promoter is expressed.

  The T7-like promoter has a sequence identity of 50% or more, 50% to 95%, 50% to 90%, 50% to 85%, or 55% to 80% with the T7 promoter sequence represented by SEQ ID NO: 4. Refers to a promoter containing a base sequence. In addition, the T7-like promoter has a nucleotide sequence having a sequence identity of 60% or more, 65% or more, 70% or more, 80% or more, 85% or more, or 90% or more with the nucleotide sequence of SEQ ID NO: 5. The promoter may include. T7-like promoters are recognized by T7-like RNA polymerase. Specific examples of the T7-like promoter are a T3 promoter, an SP6 promoter, and a promoter containing the nucleotide sequence represented by SEQ ID NO: 5.

  The target protein is not particularly limited, but is preferably a protein intended for production on an industrial scale. Examples of the target protein include a protein that can be used for industry, a protein that can be used for medical treatment, and a structural protein. Specific examples of proteins that can be used in industry or medicine include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane proteins, 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 of antibodies. Specific examples of the structural proteins include spider silk, silkworm silk, keratin, collagen, elastin, and resilin, and proteins derived therefrom.

Examples of proteins derived from spider silk or silkworm silk, which are fibroin-like proteins, include a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In Formula 1, the (A) _n motif represents an amino acid sequence composed of 4 to 20 amino acid residues, and the number of alanine residues is 80% or more of the total number of amino acid residues in the (A) _n motif. REP indicates an amino acid sequence composed of 10 to 200 amino acid residues. m shows the integer of 8-300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences. Specific examples of the protein derived from spider silk or silkworm silk include a protein containing the amino acid sequence represented by SEQ ID NO: 6 (PRT410) or SEQ ID NO: 7 (PRT918).

Examples of the collagen-derived protein include a protein containing a domain sequence represented by Formula 2: [REP2] _o . In Formula 2, o represents an integer of 5 to 300. REP2 represents an amino acid sequence composed of Gly-XY, and X and Y represent any amino acid residue other than Gly. A plurality of REP2s may have the same amino acid sequence or different amino acid sequences. As a specific example of the protein derived from collagen, a protein containing the amino acid sequence represented by SEQ ID NO: 8 can be mentioned. The amino acid sequence represented by SEQ ID NO: 8 corresponds to a repeat portion and a motif of a partial sequence of human collagen type 4 obtained from the NCBI database (Genebank accession number of NCB1: CAA56335.1, GI: 3702452). The amino acid sequence represented by SEQ ID NO: 9 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from residues 301 to 540.

As a protein derived from resilin, for example, a protein containing a domain sequence represented by Formula 3: [REP3] _p can be mentioned. In Formula 3, p represents an integer of 4 to 300. REP3 shows an amino acid sequence composed of Ser-JJ-Tyr-Gly-U-Pro. J represents any amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr. U represents any amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser. A plurality of REP3s may have the same amino acid sequence or different amino acid sequences. As a specific example of the protein derived from resilin, a protein containing the amino acid sequence represented by SEQ ID NO: 10 can be mentioned. In the amino acid sequence represented by SEQ ID NO: 10, in the amino acid sequence of resilin (Genebank Accession No. NP 611157 of NCBl, Gl: 246654243), Thr at the 87th residue is replaced with Ser, and Asn at the 95th residue is replaced with Ser. In which the amino acid sequence represented by SEQ ID NO: 9 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence obtained by substituting Asp with Asp.

  Examples of the protein derived from elastin include a protein having an amino acid sequence such as GeneBank Accession Nos. AAC98395 (human), I47076 (sheep), NP786966 (bovine) of NCBl. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 11 can be mentioned. The amino acid sequence represented by SEQ ID NO: 11 corresponds to the amino acid sequence represented by SEQ ID NO: 9 (tag sequence) at the N-terminal of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of Genebank accession number AAC98395 of NCB1. And a hinge sequence).

  Examples of keratin-derived proteins include, for example, Capra hircus type I keratin and the like. Specific examples include a protein comprising the amino acid sequence represented by SEQ ID NO: 12 (the amino acid sequence of Genebank accession number ACY30466 of NCB1).

  The nucleic acid encoding the protein of interest is under the control of a third promoter and is transcribed by the exogenous RNA polymerase expressed from the second expression cassette in the presence of maltose. In the absence of maltose, as described above, transcription of a nucleic acid encoding an exogenous RNA polymerase is suppressed. Thus, in the absence of maltose, no exogenous RNA polymerase is produced, whereby the third promoter does not function and the nucleic acid encoding the protein of interest is not transcribed.

  The one or more second operators that may be included in the third expression cassette are preferably operators in the maltose operon, and may be, for example, an operator having a base sequence represented by SEQ ID NO: 2. The third expression cassette may include more than one operator and may include three operators to facilitate binding of the repressor to the operator. The one or more second operators may be operably linked to a third promoter. The position of each operator from the transfer start point and the distance between each operator are as described above.

  If the third expression cassette includes the one or more second operators, the second repressor described below binds to the second operator in the absence of maltose. Also, if the first repressor is the same as the second repressor, the first repressor may also bind to the second operator in the absence of maltose. Binding of the first repressor or the second repressor to the second operator prevents binding of the exogenous RNA polymerase to a third promoter operably linked to the operator, thereby controlling the control of the third promoter. Transcription of the underlying nucleic acid encoding the protein of interest is suppressed. That is, by arranging an operator in the third expression cassette in addition to the second expression cassette, transcription of the nucleic acid encoding the target protein, that is, expression of the target protein can be more strictly controlled.

  The second double-stranded DNA fragment may further comprise a fourth expression cassette comprising a fourth promoter and a nucleic acid under the control of the fourth promoter and encoding a second repressor protein. The fourth promoter is not limited as long as it functions in the host cell, and any of the above-listed promoters may be used as the first promoter. The fourth promoter may be the same as the first promoter.

  As the nucleic acid encoding the second repressor, any of the nucleic acids listed above as the nucleic acid encoding the first repressor may be used. The nucleic acid encoding the second repressor may be the same as the nucleic acid encoding the first repressor.

  The third expression cassette and the fourth expression cassette can further include a sequence involved in gene expression, such as a terminator and a ribosome binding site, if necessary. The second double-stranded DNA fragment may be an expression cassette for expressing another protein, such as an expression cassette having an antibiotic resistance gene, as long as the function of the third expression cassette and the fourth expression cassette is not hindered. May be further included.

  The third expression cassette and the fourth expression cassette may be connected in a direction in which the transcription directions of the respective cassettes are the same, or may be connected in opposite directions. That is, even if the sense strand of the nucleic acid encoding the target protein and the sense strand of the nucleic acid encoding the second repressor are on the same strand among the two strands of the second double-stranded DNA fragment Or on different chains. When the second double-stranded DNA fragment is retained in a host cell as circular extrachromosomal DNA such as a plasmid, the third expression cassette and the fourth expression cassette preferably have the transcription directions of the respective cassettes reversed. Are connected in the following direction. When the transcription direction of the third expression cassette and the fourth expression cassette are the same, for example, the terminator in the third expression cassette located upstream is read-through, so that the third expression cassette in the fourth expression cassette is read out. Excessive transcription of the nucleic acid encoding the two repressors may occur. When the transcription direction of the third expression cassette and that of the fourth expression cassette are reversed, such excessive transcription of the nucleic acid encoding the second repressor can be prevented.

  Each of the first double-stranded DNA fragment and the second double-stranded DNA fragment may be an expression vector. The expression vector is not limited as long as it can replicate autonomously in the host cell or can be integrated into chromosomal DNA of the host cell, and can be appropriately selected depending on the host cell. Examples of the expression vector include a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, and an artificial chromosome vector. The first double-stranded DNA fragment that functions as an expression vector can be obtained, for example, by inserting the first expression cassette and the second expression cassette into a known vector. The second double-stranded DNA fragment that functions as an expression vector can be obtained, for example, by inserting a third expression cassette and optionally a fourth expression cassette into a known vector. However, the lacI gene and the lac operator in these known vectors are appropriately removed or replaced with the above-described repressor and operator.

  Known vectors include, for example, pUC19 (Takara Bio Inc.), pTWV228 (Takara Bio Inc.), pMW119 (Nippon Gene), CRIM plasmid vector, pBTrp2, pBTac1, pBTac2 (all manufactured by Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen), pKYP10 (JP-A-58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (Stratagene), pTrs30 (prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)), pTrs32 [Escherichia coli JM109 / pTr32] FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), JP-A-60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), No. 60-221091], pTerm2 (US Pat. No. 4,686,191, US Pat. No. 4,939,094, US Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pACYC184 (manufactured by Nippon Gene, product number 313-02201), pET system (manufactured by Novagen), YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 ( ATCC 37419), YIp, pHS19, pHS15, pA0804, pHIL3O1, pHIL-S1, pPIC9K, pPICZα, pGAPZα, pPICZB, and Autographa californica nuclei polyhedrosis virus, a virus that infects insects of the night moth family. Baculovirus expression vectors such as Autographa californica nuclei polyhedrolysis virus [Baculov rus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)].

  The copy number of the expression vector is not particularly limited, but when a cell lacking maltose utilization ability is used as a host, the copy number of the vector containing the second double-stranded DNA fragment is preferably 1 to 700, More preferably, it is 1-20, and still more preferably 1-5.

[2. Recombinant cell]
A recombinant cell according to one embodiment of the present invention is a recombinant cell including the expression system according to the above embodiment. Recombinant cells can be obtained, for example, by introducing the first double-stranded DNA fragment and the second double-stranded DNA fragment into host cells.

  As the host cell, any of prokaryotic cells such as bacteria and eukaryotic cells such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.

  Examples of prokaryotes include microorganisms belonging to the genus Escherichia, Vibrio, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like.

  Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli BL21 (Novagen), Escherichia coli BL21 (DE3) (Life Technologies), Escherichia coli BL21 (Merck Millipore), Escherichia coli BLR (DE3) ( Merck Millipore), Escherichia coli DH1, Escherichia coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC 23506), Escherichia coli KY3276, Escherichia coli MC, Escherichia coli MMC 47076), 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 110 (Escherichia coli) ATCC 27325), Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, and the like.

  Examples of the yeast include, for example, genus Saccharomyces, genus Schizosaccharomyces, genus Kluyveromyces, genus Trichosporon, genus Trichosponi, and genus Schiwanidas And yeasts belonging to the genus Yarrowia and the genus Hansenula. 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), Schwanniomyces occidentalis, Candida utilis, Pichia pastoris (Pic) hia pastoris) Pichia Angusuta (Pichia angusta), Pichia methanolica (Pichia methanolica), Pichia polymorpha (Pichia polymorpha), Pichia stipitis (Pichia stipitis), Yarrowia lipolytica (Yarrowia lipolytica), Hansenula polymorpha (Hansenula polymorpha) And the like.

  Examples of the yeast include, for example, genus Saccharomyces, genus Schizosaccharomyces, genus Kluyveromyces, genus Trichosporon, genus Trichosponi, and genus Schiwanidas And yeasts belonging to the genus Yarrowia and the genus Hansenula.

  Examples of the filamentous fungi include the genus Acremonium, the genus Aspergillus, the genus Ustilago, the genus Trichoderma, the genus Neurospora, and the genus Fusarium, Fusarium, Fusarium. The genus Penicillium, the genus Myceliophtora, the genus Botrytis, the genus Magnaporthe, the genus Mucor, the genus Metahizium, the genus Monascus R. Monascus And bacteria belonging to the genus Rhizomucor.

  As host cells, prokaryotic organisms such as Escherichia coli (Escherichia coli), Bacillus subtilis, Pseudomonas, Corynebacterium, and Lactococcus are preferred, and Escherichia coli is more preferred.

  The recombinant cell is preferably a cell that cannot assimilate maltose from the viewpoint of sustaining the induction of the expression of the target protein. Cells that cannot assimilate maltose do not have the maltose assimilation gene or have a maltose assimilation gene with a mutation. Examples of the maltose utilization gene include malQ, malP, malZ, and malS. Preferably, the recombinant cells do not have malQ.

  In the recombinant cells, the first double-stranded DNA fragment and the second double-stranded DNA fragment may be independently incorporated into chromosomal DNA, or may be retained as extrachromosomal DNA such as a plasmid. Good. Recombinant cells may have, for example, a first double-stranded DNA fragment integrated into the chromosomal DNA and a second double-stranded DNA fragment maintained as a plasmid.

  Introduction of the first double-stranded DNA fragment or the second double-stranded DNA fragment into a host cell can be performed by a known method. For example, an expression vector containing the first double-stranded DNA fragment or the second double-stranded DNA fragment can be introduced into a host cell.

  As a method for integrating the first double-stranded DNA fragment or the second double-stranded DNA fragment into the chromosome of the host cell, a known method can be used. Known methods include, for example, the λ red method, the Red / ET homologous recombination method using a recombination mechanism in the double-strand break repair of λ phage, and the transfer method using transposon activity using pUT-mini Tn5. Is mentioned.

[3. Method for producing target protein]
The method for producing a target protein according to one embodiment of the present invention includes a step of culturing the above-mentioned recombinant cell in the presence of maltose. By culturing the above recombinant cells in the presence of maltose, the expression of the target protein in the recombinant cells can be induced. The recombinant cells can be cultured, for example, in a medium supplemented with maltose. The medium is not particularly limited, and can be selected from known natural or synthetic media according to the type of the recombinant cell. The medium preferably contains inorganic salts, a nitrogen source, and a carbon source other than maltose that the recombinant cells can assimilate. The concentration of maltose in the medium may be, for example, 0.1-4 w / v%, 1-3 w / v%, or 1.5-2 w / v%. The pH of the medium may be, for example, between 3.0 and 9.0. The culture temperature is, for example, 15 to 40 ° C. The culture time may be, for example, 1 to 30 hours.

  The method for producing the target protein according to the embodiment may include, before the step of culturing the recombinant cell in the presence of maltose, a step of culturing the recombinant cell in the absence of maltose. By growing the recombinant cells to a predetermined amount before inducing the expression of the target protein, the target protein can be produced more efficiently. The medium used for the culture may be the same as the above-mentioned medium except that it does not contain maltose. The culture temperature is, for example, 15 to 40 ° C. The culture time may be, for example, 3 hours or more, and may be 5 to 15 hours.

[Preparation of spider silk protein expression vector]
First, a DNA fragment containing the malR gene of Streptococcus pneumoniae was synthesized as follows. An aqueous solution (100 pmol / μL) of the primer shown in Table 1 was mixed in an amount of 2 μL. 1 μL of this mixed aqueous solution was mixed with 25 μL of PrimeSTAR (registered trademark) Max DNA Polymerase Premix (Takara Bio Inc.). Ultrapure water was added to a final volume of 50 μL to obtain a reaction mixture. After heating the reaction mixture at 98 ° C. for 1 minute, the PCR profile of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 5 seconds was repeated for 15 cycles, and the reaction mixture was further heated at 72 ° C. for 2 minutes. Reacted. To 10 µ of the obtained PCR product, 1 µL of an aqueous solution of a primer (10 pmol / µL) shown in Table 1 and 25 µL of PrimeSTAR Max DNA Polymerase Premix were added. Ultrapure water was added to a final volume of 50 μL to obtain a reaction mixture. After heating the reaction mixture at 98 ° C. for 1 minute, the PCR profile of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 5 seconds was repeated 25 cycles, and the reaction mixture was further heated at 72 ° C. for 10 seconds. The reaction was performed for seconds. The obtained PCR product was purified. In this example, innuPREP PCR pure Kit | innuPREP PCR pure 96 Kit (Analytical Jena) was used for purification of DNA. To the purified PCR product, 1 μL of an aqueous solution (10 pmol / μL) of a primer having the nucleotide sequence represented by SEQ ID NO: 37 or SEQ ID NO: 38, 22 μL of ultrapure water, and 25 μL of PrimeSTAR Max DNA Polymerase Premix were added. PCR was performed. The PCR conditions were as follows: After heating at 98 ° C. for 1 minute, the PCR profile of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 7 seconds was repeated 30 cycles, and the reaction mixture was subjected to the reaction. The reaction was further performed at 72 ° C. for 10 seconds. The subsequent PCR was performed under the same conditions. The obtained PCR product was purified to obtain a DNA fragment 1 containing the lacI promoter, the malR gene, and the rrnBT1 terminator in this order.

  Next, PCR was performed using a vector as a template and primers having the base sequences of SEQ ID NOs: 39 and 40 as primers. As a vector, pUC19 (Takara Bio Inc.), pTWV228 (Takara Bio Inc.), or pMW119 (Nippon Gene) was used. The obtained PCR product was purified to obtain three kinds of DNA fragments 2 containing a vector sequence.

  DNA fragment 1 and DNA fragment 2 containing the vector sequence were fused using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs Japan). The resulting plasmid is E. coli. coli HST02 (Takara Bio Inc.) was transformed. The next day, plasmids were collected from the transformant colonies, and plasmids having the correct sequence were selected by sequence analysis. PCR was performed on this plasmid to obtain a DNA fragment 3, which is a vector containing the lacI promoter, the malR gene, and the rrnBT1 terminator in this order. DNA fragment 3 contains almost the entire length of the vector sequence. For the synthesis of DNA fragment 3 containing pUC19 or pMW119, a primer having the nucleotide sequence shown in SEQ ID NO: 41 and SEQ ID NO: 42 was used. For the synthesis of DNA fragment 3 containing pTWV228, primers having the nucleotide sequences of SEQ ID NO: 43 and SEQ ID NO: 44 were used.

  In addition, a DNA fragment 4 containing a T7 promoter, a lac operator, a nucleic acid encoding a spider silk protein (hereinafter also referred to as SSP), a T7 terminator, and a rrnB terminator in this order was prepared. As the nucleic acid encoding the SSP, a nucleic acid encoding an SSP having the amino acid sequence represented by SEQ ID NO: 7 was used.

  DNA fragment 3 and DNA fragment 4 were fused by NEBuilder HiFi DNA Assembly Master Mix. The resulting three plasmids were used for E. coli. coli HST02 was transformed. The next day, plasmids were collected from the transformant colonies, and plasmids having the correct sequence were selected by sequence analysis.

  Next, the lac operator in the above plasmid was replaced with the operator having the nucleotide sequence of SEQ ID NO: 2 (Str-binding site) by the following method. PCR was performed using a plasmid as a template and primers having the nucleotide sequences of SEQ ID NOs: 45 and 46 as primers. The resulting PCR product was treated with DpnI. The resulting three plasmids were used for E. coli. coli HST02 was transformed. The next day, plasmids were collected from the transformant colonies, and plasmids having the correct sequence were selected by sequence analysis. FIGS. 2 to 4 show vector maps of the obtained SSP expression vectors.

[Preparation of T7 RNA polymerase expression vector]
Using the primers shown in Table 2, PCR was repeated in the same manner as in the synthesis of DNA fragment 1 to obtain DNA fragment 5. DNA fragment 5 contains a lacUV5 promoter and three operators (Str-binding sites) each having a base sequence represented by SEQ ID NO: 2.

  In addition, PCR was performed using DNA fragment 1 as a template and primers having the nucleotide sequences of SEQ ID NOS: 59 and 60 to obtain DNA fragment 6. DNA fragment 6 contains the lacI promoter and the malR gene.

  Further, PCR was performed using the vector (attlambda-Cm2-Tsp-lac-T7P) shown in FIG. 5 as a template and primers having the nucleotide sequences shown in SEQ ID NO: 61 and SEQ ID NO: 62 to obtain DNA fragment 7. . The vector shown in FIG. 5 is a CRIM plasmid (Haldimann and Wanner (“Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-functionstudies of bacteria,” J Bacteriology, 183: 6384-6393 (2001)). ) Is a backbone plasmid. DNA fragment 7 is a fragment obtained by removing a partial sequence from atlambda-Cm2-Tsp-lac-T7P, and contains a gene encoding T7 RNA polymerase.

  DNA fragments 5, 6, and 7 were fused with NEBuilder HiFi DNA Assembly Master Mix to obtain a T7 RNA polymerase expression vector shown in FIG. The T7 RNA polymerase expression vector also contains a chloramphenicol (Cm) resistance gene.

[Transformation]
The T7 RNA polymerase expression vector was prepared using the helper plasmid pAH69 (see US Patent Application Publication No. 2004/0033608) in J Bacteriol. 2001 Nov; 183 (21): 6384-6393. E. coli HST02 was inserted into the attHK022 site. Transformants incorporating a single plasmid were selected on plates containing chloramphenicol.

  The malQ gene of the above transformant was disrupted by the method described in PNAS June 6, 200.97 (12) 6640-6645. Thereafter, the obtained malQ-disrupted strain was transformed with an SSP expression vector to obtain 19, 228, and 119 strains without the malQ gene. The 19 strains, 228 strains and 119 strains were obtained from SSP expression vectors derived from pUC19 (copy number: 500 to 700), pTWV228 (copy number: 15 to 20), and pMW119 (copy number: 5 or less), respectively. Have.

[Expression induction]
Using 10 mL of LB medium containing ampicillin, 19, 228, and 119 strains without the malQ gene were cultured from colonies. When the OD exceeded 1, the culture was divided into two tubes of 5 mL each, and glucose or maltose was added to the culture so that the final concentration was 1.5% (w / v). Table 3 shows the OD600 nm value of the culture solution before the addition of the sugar and 2 hours after the addition of the sugar. Two hours after the sugar was added, the cells were collected and subjected to SDS-PAGE. FIG. 7 shows an SDS-PAGE electrophoresis photograph. In FIG. 7, "G" and "M" represent glucose and maltose, respectively.

  The same experiment as described above was carried out using 19 strains, 228 strains and 119 strains having the malQ gene. FIG. 8 shows an SDS-PAGE electrophoresis photograph. In FIG. 8, “G” and “M” represent glucose and maltose, respectively.

  As shown in Table 3, the malQ-disrupted strain had normal growth ability. In addition, as shown in FIGS. 7 and 8, expression of the SSP gene was significantly induced by the addition of maltose regardless of the presence or absence of the malQ gene. On the other hand, in the malQ-disrupted strain, stronger expression induction was observed in the strain having the low-copy-number plasmid (FIG. 7), whereas in the malQ-strain, stronger expression was observed in the strain having the high-copy-number plasmid. Induction was observed (FIG. 8).

Claims (13)

An expression system, comprising: a first double-stranded DNA fragment containing a first expression cassette and a second expression cassette; and a second double-stranded DNA fragment containing a third expression cassette,
The first expression cassette comprises a first promoter, and a nucleic acid under the control of the first promoter and encoding a first repressor protein,
The second expression cassette comprises a second promoter, one or more first operators, and a nucleic acid under the control of the second promoter and encoding an RNA polymerase;
The third expression cassette includes a third promoter and a nucleic acid that is under the control of the third promoter and encodes a target protein, wherein the third promoter is recognized by the RNA polymerase;
The first repressor protein binds to the one or more first operators in the absence of maltose and represses transcription of the nucleic acid encoding the RNA polymerase, but in the presence of maltose, An expression system that does not bind to more than one primary operator.   The expression system according to claim 1, wherein the first repressor protein is a MalR protein.   The expression system according to claim 1 or 2, wherein the nucleic acid encoding the first repressor protein has a base sequence represented by SEQ ID NO: 1.   The expression system according to any one of claims 1 to 3, wherein the one or more first operators are operators having a base sequence represented by SEQ ID NO: 2.   The expression system according to any one of claims 1 to 4, wherein the first expression cassette includes two or more of the first operators.   The expression system according to any one of claims 1 to 5, wherein the RNA polymerase is a T7 RNA polymerase, and the third promoter is a T7 promoter. Wherein the second double-stranded DNA fragment further comprises a fourth expression cassette comprising a fourth promoter and a nucleic acid under the control of the fourth promoter and encoding a second repressor protein;
Said third expression cassette further comprises one or more second operators;
The second repressor protein binds to the one or more second operators in the absence of maltose and represses transcription of a nucleic acid encoding the protein of interest, but in the presence of maltose, The expression system according to any one of claims 1 to 6, wherein the expression system does not bind to one or more second operators. An expression system according to any one of claims 1 to 7,
A recombinant cell wherein the RNA polymerase is exogenous.   The recombinant cell according to claim 8, wherein the recombinant cell is Escherichia coli.   The recombinant cell according to claim 8, wherein the recombinant cell is unable to utilize maltose.   The recombinant cell according to any one of claims 8 to 10, wherein the first double-stranded DNA fragment is contained in chromosomal DNA, and the second double-stranded DNA fragment is contained as extrachromosomal DNA.   The recombinant cell according to claim 11, wherein the number of copies of the extrachromosomal DNA is 1 to 700.   A method for producing a target protein, comprising a step of culturing the recombinant cell according to any one of claims 8 to 12 in the presence of maltose.