JP2020014392A - Expression vectors for prokaryotes - Google Patents

Abstract

To provide expression vectors for prokaryotes designed to include a translation enhancing sequence in an untranslated region of a polynucleotide encoding a protein to be expressed.SOLUTION: Disclosed herein is an expression vector for prokaryotes comprising a polynucleotide described in (1) or (2), (1) a polynucleotide being an entire or partial sequence of a polynucleotide encoding a myosin regulatory light chain (mlcR) which comprises specific 483 bases, where the partial sequence comprises at least a base sequence from the positions 474 to 483, (2) a polynucleotide having at least 90% sequence identity to the polynucleotide (1), a polynucleotide encoding a ribosome-binding site and a polynucleotide encoding a protein to be expressed in this order.SELECTED DRAWING: None

Description

  The present invention relates to a prokaryotic expression vector, a prokaryote containing such a prokaryotic expression vector, and a method for producing a protein to be expressed using such a prokaryote.

  With the development of genetic recombination technology in recent years, the production of various useful proteins using microorganisms has come to be performed. Above all, bacteria are widely used to produce useful substances such as amino acids and proteins. In particular, in recent years, a technique has been known in which a gene for a pharmaceutically or industrially useful protein is introduced into a bacterium to transform the bacterium, and the transformant is used to efficiently produce a useful protein.

  Escherichia coli, a kind of Gram-negative bacteria, is widely used as a bacterium used for protein expression. The technology using Escherichia coli for protein expression has been widely used for the production of industrial proteins, food processing proteins and pharmaceutical proteins.

  Various expression vectors have been developed for protein production using Escherichia coli. The most widely used expression system vector is the pET expression system vector (see Non-Patent Document 1). The pET expression system vector is a vector that expresses a target gene cloned downstream of the T7 promoter using a very strong T7 RNA polymerase. However, there have been reports of proteins that are not versatile and cannot be successfully expressed. Also, pCold expression system vectors (see Non-Patent Documents 2 and 3) are widely used. This pCold expression system vector is different from the pET expression system vector using the T7 promoter in that the target gene is expressed at a low temperature using the cspA promoter. Further, in the pCold expression system vector, translation is promoted by using a translation enhancing element (TEE) to increase the amount of synthesis. However, since this TEE is located downstream of the initiation codon of the target gene, it becomes a so-called "tag" and is expressed as a fusion protein with the protein to be expressed. As a result, an adverse effect of losing native protein functions is also assumed.

Rosenberg, AH, Lade, BN, Dao-shan, C., Lin, S.-W., Dunn, JJ, & Studier, FW (1987) .Vectors for selective expression of cloned DNAs by T7 RNA polymerase.Gene, 56 (1), 125-135. Qing, G., Ma, LC, Khorchid, A., Swapna, GVT, Mal, TK, Takayama, MM, et al. (2004) .Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol, 22 (7), 877-882. Etchegaray, J.P., & Inouye, M. (1999) .Translational enhancement by an element downstream of the initiation codon in Escherichia coli.J Biol Chem, 274 (15), 10079-10085.

  As described above, the expression may not be sufficient in the pET expression system vector, and in the pCold expression system vector, the TEE is located downstream of the initiation codon of the target gene, so that the target expression protein and TEE remain fused. was there. Accordingly, an object of the present invention is to provide a prokaryotic expression vector designed to include a sequence having a translation promoting effect in a non-translated region of a polynucleotide encoding a protein to be expressed.

  The present inventors have focused on the myosin regulatory light chain (mlcR) gene of cellular slime mold, and together with a polynucleotide encoding the mlcR gene, a polysaccharide encoding a ribosome binding site (RBS). When Escherichia coli was transformed by incorporating the nucleotides and the polynucleotide encoding the mEGFP gene into a commonly used pUC vector, the plasmids were cultured in a medium without a transcription promoter such as isopropyl-β-thiogalactopyranoside (IPTG). The inventors found that GFP was highly expressed in the transformants, and completed the present invention.

That is, the present invention is as follows.
[1] A prokaryotic expression vector comprising, in order, a polynucleotide according to the following (1) or (2), a polynucleotide encoding a ribosome binding site, and a polynucleotide encoding a protein to be expressed.
(1) The full length or partial sequence of a polynucleotide encoding the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1, which is at least the 474-483rd base of the nucleotide sequence shown in SEQ ID NO: 1. A polynucleotide comprising the sequence;
(2) a polynucleotide having at least 90% or more sequence identity with the polynucleotide of (1);
[2] The prokaryotic expression vector according to the above [1], comprising a 4-20 base spacer sequence between the polynucleotide encoding the ribosome binding site and the polynucleotide encoding the protein to be expressed. .
[3] The prokaryotic expression vector according to the above [1] or [2], wherein at least the base sequence at positions 474 to 483 of the base sequence shown in SEQ ID NO: 1 is the base sequence shown in SEQ ID NO: 2. .
[4] The expression vector for prokaryote according to any one of [1] to [3], wherein the prokaryote is Escherichia coli.
[5] A prokaryote containing the expression vector for prokaryote according to any one of [1] to [4].
[6] A method for producing a protein, comprising a step of culturing the prokaryote according to the above [5] and a step of collecting a protein to be expressed from the cultured prokaryote.
[7] A kit for expressing a protein, the kit comprising the prokaryotic expression vector according to any one of the above [1] to [4] and instructions for expressing the protein.

  According to the present invention, the expression level of a protein to be expressed can be increased without adding a mutation.

It is a figure which showed the positional relationship of a mlcR gene and a forward primer and a reverse primer in the manufacturing process of plasmid vector "mlcR-RBS-Spacer-GFP-pUC19". It is a figure which shows the map of a plasmid vector "mlcR-RBS-Spacer-GFP-pUC19". In Example 1, Escherichia coli transformed with the plasmid vectors "mlcR-RBS-Spacer-GFP-pUC19" and "GFP-pUC19" was applied to an LB agar medium, and cultured at 37 ° C overnight in a dark environment. It is a photograph taken under blue LED irradiation. FIG. 4 is a diagram showing the results of observing a culture solution with a fluorescence microscope after culturing a colony confirmed to emit green fluorescence in an LB medium overnight in a dark environment in Example 1. FIG. 4 is a diagram showing the results obtained by culturing colonies confirmed to emit red fluorescence in an LB medium in a dark environment overnight in Example 1 and then observing the culture solution with a fluorescence microscope. In Example 2, (a) is a diagram showing the mlcR-deficient site, and (b) is a result of culturing the defective mlcR-GFP transformant and observing the fluorescence intensity (Z score) of GFP with a fluorescence microscope. FIG. FIG. 7 shows the results of comparison of the growth rate of bacteria and the yield of plasmid vectors with and without GFP expression in Example 3. The horizontal axis in (a) represents time, and the vertical axis represents the relative cell concentration when the cell concentration at the start of growth is set to 1. The vertical axis in (b) shows the yield of the plasmid vector. In Example 4, it is a figure which shows the result of having investigated four types of vector into Escherichia coli, and having investigated the GFP fluorescence Z score when it culture | cultivated in the culture medium with or without glucose. In Example 5, it is a figure which shows the result of having investigated four types of vector into Escherichia coli, and having investigated the GFP fluorescence Z score when it culture | cultivated in the culture medium with or without IPTG.

  The vector according to the present invention is (1) the full-length or partial sequence of the polynucleotide encoding the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1, which is at least 474 to 483 of the nucleotide sequence shown in SEQ ID NO: 1. A polynucleotide comprising a base sequence; a prokaryotic expression vector (hereinafter, referred to as a polynucleotide) comprising the polynucleotide according to (1) or (2), a polynucleotide encoding a ribosome binding site, and a polynucleotide encoding a protein in order. If such a vector is used, a sequence having a translation promoting effect is located on the 5 ′ side (upstream) of a start codon in a polynucleotide encoding a protein. It is possible to express a protein to be expressed without translating a certain sequence That.

  The myosin regulatory light chain (mlcR) in the present vector is a component of myosin II, a motor protein that moves on actin. Such myosin regulatory light chains are known to be involved in functions such as cell division, cell motility, and cell morphology changes.

  The myosin regulatory light chain may be derived from Dictyostelium discoideum, Escherichia coli (Physarum polycephalum), ringworms such as flagellate, sponges, nematodes, insects such as Drosophila, and tail cables such as sea squirts. Animals, hemichords such as gibberworms, echinoderms such as sea urchins, caudal animals such as slugs, fish such as zebrafish, amphibians such as Xenopus laevis, birds such as chickens, humans, mice, rats, guinea pigs, rabbits and cats And mammals such as dogs, horses, cows, monkeys, sheep, goats and pigs, and cellular slime molds are preferred.

  The polynucleotide encoding the myosin regulatory light chain (mlcR) can be appropriately obtained by searching publicly known documents and databases such as NCBI (http://www.ncbi.nlm.nih.gov/guide/). . As the polynucleotide encoding the full length of the myosin regulatory light chain (mlcR) derived from cellular slime mold, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 can be mentioned.

The partial sequence of the polynucleotide encoding the myosin regulatory light chain (mlcR) means a sequence in which a part of the polynucleotide encoding the full length of the myosin regulatory light chain (mlcR) is deleted. The partial sequence of the polynucleotide encoding the myosin regulatory light chain (mlcR) includes:
The base sequence shown in SEQ ID NO: 2 (the 474th to 483rd 10 bases in the base sequence shown in SEQ ID NO: 1),
The nucleotide sequence shown in SEQ ID NO: 3 (the 20th nucleotide at positions 464 to 483 in the nucleotide sequence shown in SEQ ID NO: 1),
The base sequence shown in SEQ ID NO: 4 (25th bases at positions 459 to 483 of the base sequence shown in SEQ ID NO: 1),
The base sequence shown in SEQ ID NO: 5 (the 454th to 483rd 30 bases in the base sequence shown in SEQ ID NO: 1),
The nucleotide sequence shown in SEQ ID NO: 6 (the 440th to 483rd nucleotides of the nucleotide sequence shown in SEQ ID NO: 1),
The base sequence shown in SEQ ID NO: 7 (the 439th to 483rd 45 bases in the base sequence shown in SEQ ID NO: 1),
The nucleotide sequence shown in SEQ ID NO: 8 (the 424-483rd 60 nucleotides in the nucleotide sequence shown in SEQ ID NO: 1),
The nucleotide sequence shown in SEQ ID NO: 9 (74 nucleotides at positions 410 to 483 of the nucleotide sequence shown in SEQ ID NO: 1),
The nucleotide sequence shown in SEQ ID NO: 10 (110 to 374th to 483rd nucleotides in the nucleotide sequence shown in SEQ ID NO: 1),
The base sequence shown in SEQ ID NO: 11 (the 235th to 243rd bases in the base sequence shown in SEQ ID NO: 1),
The base sequence shown in SEQ ID NO: 12 (the 219-483rd 265 bases in the base sequence shown in SEQ ID NO: 1),
A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13 (355th nucleotide at positions 129 to 483 of the nucleotide sequence of SEQ ID NO: 1) can be mentioned.

  In the present specification, "a full-length or partial sequence of a polynucleotide encoding the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1, wherein at least the base sequence at positions 474 to 483 of the base sequence shown in SEQ ID NO: 1 is "Includes" refers to at least the 474th to 483rd base sequences of the base sequence shown in SEQ ID NO: 1, that is, the 3 'terminal 10 bases in the polynucleotide encoding the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1. It means that it is necessary to include it. Specifically, the polynucleotide encoding the full length of the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1 may be used, or the 3 ′ terminal side of the polynucleotide encoding the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1 May be a polynucleotide encoding a partial sequence in which any base at the 5 'end of the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1 has been deleted.

  As used herein, “at least 90% or more sequence identity” means that the sequence identity is 90% or more, preferably 93% or more, more preferably 95% or more, and even more preferably 98% or more. Above, even more preferably above 99%, most preferably 100% sequence identity.

  The "base sequence having at least 90% or more sequence identity with the polynucleotide according to (1)" is, in other words, "in the polynucleotide according to (1), one or several bases are deleted, It is a nucleotide sequence having a function equivalent to that of the polynucleotide described in (1) above, which is a "substituted, inserted, and / or added polynucleotide". Here, “polynucleotide in which one or several base sequences are deleted, substituted, inserted, and / or added” can be appropriately adjusted according to the length of the polynucleotide. Within the range, preferably within the range of 1 to 20, more preferably within the range of 1 to 15, more preferably within the range of 1 to 10, more preferably within the range of 1 to 5, and even more preferably 1 A polynucleotide in which a number of polynucleotides in the range of 範 囲 3, more preferably in the range of ２1 to 2, is deleted, substituted, inserted and / or added. Mutation treatment of these polynucleotides can be performed by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis.

  The polynucleotide encoding the ribosome binding site (RBS) in the present vector is located about 3 to 9 bases upstream of the translation initiation codon (AUG, GUG, UUG, AUU, CUG) in prokaryotic mRNA. A polynucleotide rich in purine bases (adenine (A), guanine (G)), a site that helps recruit ribosomes to mRNA and initiates protein synthesis by placing the ribosome at the start codon. The polynucleotide encoding the ribosome binding site can be designed based on the Shine-Dalgarno sequence. Specific examples include polynucleotides consisting of 4 to 9 bases rich in purine bases (adenine (A), guanine (G)), and GGAG, AGGA, GGAGG, AGGAG, GGAGGT, AGGGAG, GAGGAG, AGGAGG, AGGAGGT, AGAGGAGA and the like.

  The activity of any purine base (adenine (A), guanine (G))-rich sequence that functions as a ribosome binding site can be tested using any suitable method. For example, expression can be measured by measuring the activity of a reporter protein encoded by mRNA, as described in Example 1 of WO 2004/046321.

  It is preferable to include a spacer sequence consisting of an arbitrary base sequence between the polynucleotide encoding the ribosome binding site and the polynucleotide encoding the protein to be expressed. The spacer sequence is a polynucleotide consisting of 4 to 20 bases, preferably 5 to 11 bases, more preferably 6 to 10 bases. Such a sequence encodes a polynucleotide encoding a ribosome binding site and a protein to be expressed. The start codons of the polynucleotides are located at an appropriate distance so that the translation control function can be exerted. The base of the spacer sequence is not particularly limited, and may be any of adenine, guanine, cytosine, and thymine.

  The protein to be expressed in the present specification may be a target protein to be expressed in prokaryotes, and may be a full-length protein or a protein consisting of a partial amino acid sequence thereof depending on the use. Further, the polynucleotide encoding the protein to be expressed may be modified so that the expression is optimized in the host cell.

  Prokaryotes in the present specification may be any eubacteria or Bacillus subtilis, and examples of eubacteria include Gram-negative bacteria such as Escherichia coli and Gram-positive bacteria such as Bacillus subtilis, and preferably Escherichia coli. Can be.

  Examples of the vector used for the prokaryotic expression vector in the present specification include a plasmid vector, a fosmid, a cosmid, and a BAC (bacterial artificial chromosome) vector, and a plasmid vector is preferable. Examples of the plasmid vector include a plasmid vector that can be replicated in Escherichia coli such as pUC18, pUC19, pBR322, pBR325, pGEM3, and pGEM4, and a plasmid vector that can be replicated in Bacillus subtilis such as pUB110, pE194, pTP5, and pC194.

  In addition, the above-mentioned vector includes a promoter, a replication origin, an operator, a drug resistance gene, a multiple cloning site, a catabolic activator protein binding site (Catabolite activator protein), a signal peptide, a tag peptide, and the like, as long as the native protein function can be maintained. Alternatively, it may include a polypeptide encoding a peptide having a protease recognition site.

  Examples of the above promoter include T7 promoter, lac promoter, trp promoter, PL promoter, PR promoter, PSE promoter, SP01 promoter, SP02 promoter, penP promoter and the like. Also, artificially designed and modified promoters such as a promoter in which two P trps are connected in series (P trp × 2), a tac promoter, a let1 promoter, and a lacT7 promoter can be used.

  The replication origin may be appropriately selected according to the host cell to be used. When Escherichia coli is used as the host cell, the replication origin derived from pBR322 can be mentioned.

  The above operator may be appropriately selected according to the host cell to be used. When Escherichia coli is used as the host cell, a lac operator can be mentioned.

  Examples of the drug resistance gene include an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a neomycin resistance gene, a kanamycin resistance gene, and the like.

  Examples of the signal peptide include a Golgi translocation signal peptide, a cell membrane translocation signal peptide, a mitochondrial translocation signal peptide, a nuclear translocation signal peptide, a synaptic translocation signal peptide, a nucleolus translocation signal peptide, a nuclear membrane translocation signal peptide, a peroxisome translocation signal peptide, and the like. Can be mentioned.

  Examples of the tag peptide include DDDDK (FLAG) tag, HA tag, MYC tag, PA tag, HIS tag, V5 tag, CBD tag, CBP tag, TARGET tag, GFP tag, MBP tag, GST tag, Thioredoxin tag, and SNAP tag. , ACP tag, MCP tag, CLIP tag, TAP tag, and the like.

  The prokaryote containing the present vector can be prepared by introducing the present vector into a prokaryote, and the method for introducing the present vector into a prokaryote is not particularly limited, and a calcium phosphate method, an electroporation method, etc. Known methods can be mentioned.

  The method for producing a protein in the present specification may be any method for producing a protein, including a step of culturing a prokaryote containing the present vector and a step of recovering a protein to be expressed from the cultivated prokaryote. As the method, a known method used for ordinary culturing of prokaryotes can be used. For example, when the prokaryote is Escherichia coli, it can be cultured at about 37 ° C. and under aerobic conditions such as shaking culture or aeration-agitation culture. Examples of a method for recovering the protein from the culture solution include a known protein recovery method, for example, a method for recovering the protein by centrifugation, followed by chromatography such as gel filtration, ion exchange, and affinity. In the case of a protein secreted extracellularly or a protein having an extracellular secretion signal, it is preferable to recover the protein without destroying the cell.

  As a medium for culturing the prokaryote, a carbon source that can be used by the prokaryote, a nitrogen source, a mineral medium containing inorganic salts, and the like can be used. Is also good. Examples of carbon sources that can be used in the above-mentioned medium include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Examples of the nitrogen source that can be used in the above-described medium include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, and corn steep liquor. And substances containing a nitrogen source such as various amino acids. Examples of the inorganic substance that can be used in the above-described medium include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. it can.

  The kit in the present specification is a kit for expressing a protein, and may be any kit that includes the present vector and instructions for expressing the protein (hereinafter, also referred to as “the present kit”).

  The kit may further contain, for example, sterilized water, physiological saline, a surfactant, a buffer, a preservative, a transformation reagent, and the like, if necessary, in addition to the vector and the above instructions.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the technical scope of the present invention is not limited to these exemplifications. In addition, culture of Escherichia coli, preparation of a plasmid vector, observation with a fluorescence microscope, and measurement of proliferation of Escherichia coli were performed according to the following procedures. In addition, as a protein to be expressed, mEGFP or mRuby3, whose expression can be observed by fluorescence, was selected and used.

[Culture of E. coli]
Escherichia coli used was KST-derived strain HST08 (Takara Bio Inc.) or BL21 (DE3) (Cosmo Bio Inc.). As the LB medium, a commercially available LB medium (Nippon Genetics) was used. The LB agar medium was prepared by adding commercially available agar (Wako Pure Chemical Industries, Ltd.) to the LB medium. The culture was performed at 37 ° C. in a dark environment.

[Preparation of plasmid vector]
(1) Preparation of GFP Expression Plasmid Vector “GFP-pUC19” A plasmid vector GFP-pUC19kan (Fassmac) into which a gene enhanced monofluorescent protein (mEGFP) has been inserted will be referred to as “GFP”. PCR (polymerase chain reaction) was performed using the following primers to amplify the mEGFP gene.
Forward primer SEQ ID NO: 14
GFP_FOR: GGTCTAGAGATCT ATG GTTAGTAAAGGAG
Reverse primer SEQ ID NO: 15
GFP_REV: GCCCGGATCC CTA CTTATACA

  The obtained PCR amplification product was treated with restriction enzymes XbaI ("5'-TCTAGA-3 '") and BamHI ("5'-GGATCC-3'"), inserted into the pUC19 vector (Takara Bio Inc.), and The vector "GFP-pUC19" was prepared. The forward primer GFP_FOR contains an XbaI site, a BglII site and a start codon of GFP (underline: ATG), and the reverse primer GFP_REV contains a BamHI site and a stop codon (underline: CTA).

(2) Preparation of mlcR, RBS, Spacer, and GFP Expression Plasmid Vector “mlcR-RBS-Spacer-GFP-pUC19” (2-1) Amplification of mlcR Gene Cellularity prepared by the present inventors based on a known method Using the slime mold cDNA library as a template, the mlcR gene was amplified by PCR using the thermostable DNA replication enzyme PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). At this time, PCR was performed in two steps using two types of reverse primers to increase the amplification efficiency. In the first PCR, a forward primer containing restriction sites XbaI and BglII at the 5 ′ end (SEQ ID NO: 16: mlcR-FW 5′-GGTCTAGAGATCTATGGCCTCAAC-3 ′, and a part of RBS and a part of the spacer at the 3 ′ end) A reverse primer (SEQ ID NO: 17: mlcR-RV1 5′-CCACCCTCCTTTCTTACTGAAGAG-3 ′) containing the complementary sequence of the following sequence was designed and used: The second PCR was performed using the forward primer (SEQ ID NO: 16: mlcR-FW) And a reverse primer (SEQ ID NO: 18: mlcR-RV2 5'-TAGGATCCACCACCCTCCTTTC-3 ') containing RBS, the entire sequence of the spacer and the complementary sequence of the BamHI cleavage site on the 3' end side was designed and used. Above Forward primer, a diagram illustrating the positional relation of the reverse primer shown in Figure 1.

(2-2) Preparation of mlcR, RBS, spacer and GFP expression plasmid vector The amplification product of the mlcR gene obtained above was subjected to agarose gel electrophoresis, separated and purified, and then digested with BglII and BamHI. Similarly, the plasmid vector "GFP-pUC19" digested with BglII was ligated to prepare a plasmid vector "mlcR480G-RBS-Spacer-GFP-pUC19". Furthermore, the above plasmid vector "mlcR480G-RBS-Spacer-GFP-pUC19" was introduced into HST08 (Takara Bio Inc.) derived from Escherichia coli K-12, and the cells were transformed by ampicillin-containing LB agar medium. The obtained transformant was further cultured in an LB medium containing ampicillin, and then a plasmid vector "mlcR480G-RBS-Spacer-GFP-pUC19" was purified using a commercially available plasmid vector purification kit: FastGene Plasmid Mini (Nippon Genetics).

  The 6th to 8th bases of mlcR-RV1 shown in SEQ ID NO: 17 and the 12th to 17th bases of mlcR-RV2 shown in SEQ ID NO: 18 are complementary sequences of RBS (ribosome binding site), SEQ ID NO: 17 Are the complementary sequences of a part of the spacer sequence, and the 3rd to 11th bases of the mlcR-RV2 shown in SEQ ID NO: 18 are the complementary sequence of the spacer sequence.

  Here, by using the above-mentioned mlcR-RV1 and mlcR-RV2 primers, a base substitution (adenine (A) is originally guanine (G)) occurs at position 480 of the polynucleotide encoding mlcR shown in SEQ ID NO: 1. Therefore, in order to correct the generated base substitution (correct guanine (G) to adenine (A)), a forward primer: mlcR-repair-FW (SEQ ID NO: 19: 5′-TTCAGTAAAAAAGGAGGTGGGTGGAT-3 ′) and a reverse primer: Amplification was carried out by inverse PCR using mlcR-repair-RV (SEQ ID NO: 20: 5'-TCCTTTTTTTACTGAAGAGAGTATTAACGAAAAG-3 '). The amplification product was introduced into HST08 (Takara Bio Inc.) derived from Escherichia coli K-12 and cultured on an LB agar medium containing ampicillin for transformation. After the obtained transformant was further cultured in an LB medium containing ampicillin, the plasmid vector was purified in the same manner as described above to obtain a plasmid vector "mlcR-RBS-Spacer-GFP-pUC19". FIG. 2 shows a map of the obtained plasmid vector. In FIG. 2, CAP indicates a catabolite activator protein binding site, Plac indicates a lac promoter, and lacO indicates a lac operator.

(3) Preparation of RBS, Spacer, and GFP Expression Plasmid Vector “RBS-Spacer-pUC19” As a control without mlcR, a plasmid encoding mlcR was prepared using the above plasmid vector “mlcR-RBS-Spacer-GFP-pUC19” as a template. Using a forward primer containing guanine (G) further 3 ′ to the 3 ′ end of the nucleotide: RBS-FW (SEQ ID NO: 21: 5′-ATTCTAGAGGAGGTGGTGGGATC-3 ′) and the reverse primer: GFP_REV (SEQ ID NO: 15) After amplification by PCR, the plasmid was inserted into pUC19 treated with restriction enzymes (XbaI, BamHI) to prepare a plasmid vector "RBS-Spacer-pUC19".

(4) Preparation of defective mlcR-GFP chimeric gene Using the plasmid vector “mlcR-RBS-Spacer-GFP-pUC19” prepared in the above (2) as a template, the 3 ′ end (not including the stop codon) of the full length 483 bp of the mlcR gene ) To 10, 20, 25, 30, 40, 45, 60, 74, 110, 235, 265, 355 bp were amplified by PCR to delete a predetermined region from the 5 ′ end of the mlcR gene. Deficient mlcR-GFP chimeric gene fragments (in order, respectively, mlcR_R10, mlcR_R20, mlcR_R25, mlcR_R30, mlcR_R40, mlcR_R45, mlcR_R60, mlcR_R74, mlcR_R110, mlcR_R235, mlcR_R265 and 5cR_R were prepared) Primers used for PCR were designed to include an XbaI cleavage site at the 5 ′ end of the amplification product and a BamHI cleavage site at the 3 ′ end.

  The obtained partial base sequence of mlcR was digested with XbaI and BamHI, and then ligated with pUC19 treated with the same XbaI and BamHI, thereby obtaining the sequence in the plasmid vector “mlcR-RBS-Spacer-GFP-pUC19”. A plasmid vector “mlcR — 10-RBS-Spacer-GFP-pUC19” in which the full length (full) of mlcR shown in No. 1 was replaced with a partial base sequence (SEQ ID NOs: 2 to 13) in which a predetermined region was deleted from the 5 ′ end of mlcR. "MlcR_20-RBS-Spacer-GFP-pUC19" "mlcR_25-RBS-Spacer-GFP-pUC19" "mlcR_30-RBS-Spacer-GFP-pUC19" "mlcR_40-RBS-Spa" er-GFP-pUC19 "" mlcR_45-RBS-Spacer-GFP-pUC19 "" mlcR_60-RBS-Spacer-GFP-pUC19 "" mlcR_74-RBS-Spacer-GFP-pUC19 "" mlcR_110-RBS-SpaceC-GFP-GFP- "MlcR_235-RBS-Spacer-GFP-pUC19", "mlcR_265-RBS-Spacer-GFP-pUC19" and "mlcR_355-RBS-Spacer-GFP-pUC19" were prepared.

(5) Construction of mlcR, RBS, and mRuby3 expression plasmid vector "mlcR-RBS-Spacer-mRuby-pUC19" Instead of the mEGFP gene, the mRuby3 gene (Bajar, BT et al., 2016. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci Rep, 6, p.20889.), but using the same method as described above to prepare “mlcR-RBS-Spacer-mRuby-pUC19”. .

[Fluorescence microscope observation]
5 μL of Escherichia coli culture solution was dropped onto a cover glass (24 × 60 mm: Matsunami Glass Industry), a 1.5% agarose piece (8 × 8 × 1 mm) was put thereon, and further a cover glass (18 × 18 mm: Matsunami Glass Industry) ) And observed using an inverted fluorescence microscope (Nikon) equipped with a mercury lamp as an excitation light source. The Z score of mEGFP or mRuby3 fluorescence was calculated as follows. In addition, the fluorescence value of Escherichia coli used any one place in Escherichia coli as a representative value. As the background fluorescence, an arbitrary portion where Escherichia coli was not observed on the cover glass was used.

Z score = (fluorescence value of Escherichia coli−average value of background fluorescence at 10 places) / (standard deviation of background fluorescence at 10 places)

[Escherichia coli proliferation measurement]
The absorbance at 600 nm was measured using a spectrophotometer (Shimadzu Corporation).

[Example 1] Observation of transformant Escherichia coli (mlcR-GFP / pUC19 strain) transformed with the above plasmid vector "mlcR-RBS-Spacer-GFP-pUC19", and the above plasmid vector "GFP-pUC19" as a control Transformed Escherichia coli (GFP / pUC19 strain) was spread on an LB agar medium, cultured overnight at 37 ° C. in a dark environment, and photographed under blue LED irradiation is shown in FIG. In FIG. 3, the upper left is the GFP / pUC19 strain, and the lower right is the mlcR-GFP / pUC19 strain.

  Furthermore, a colony confirmed to emit green fluorescence was added to the culture tube to which the LB medium had been added, and after culturing overnight in a dark environment, the E. coli culture was observed by the above-mentioned fluorescence microscope observation method. Is shown in FIG.

  3 and 4, it was confirmed that the mlcR-GFP / pUC19 strain constantly emits green fluorescence only by culturing in the LB medium. Therefore, it was clarified that GFP can be expressed only by placing the polynucleotide encoding the mlcR gene and the RBS upstream of the polynucleotide encoding GFP.

  In the case where E. coli was transformed with the above-mentioned "mlcR480G-RBS-Spacer-GFP-pUC19" instead of the above-mentioned "mlcR-RBS-Spacer-GFP-pUC19", similarly, green fluorescence was constantly emitted, and GFP was also emitted. Was confirmed to be expressed (not shown). That is, it was confirmed that even a sequence in which a part of the nucleotide sequence encoding mlcR was mutated emitted green fluorescence, and had no effect on the expression of GFP.

  Furthermore, fluorescence microscopy was performed in the same manner as described above, except that E. coli was transformed with “mlcR-RBS-Spacer-mRuby-pUC19” instead of “mlcR-RBS-Spacer-GFP-pUC19”. . FIG. 5 shows the results. As shown in FIG. 5, it is clear that it is possible to emit red fluorescence and express mRuby3 only by arranging the mlcR gene and the polynucleotide encoding RBS upstream of the polynucleotide encoding mRuby3. It became.

  In addition, regardless of whether GFP or mRuby is expressed as described above, the polynucleotide encoding the mlcR gene and RBS having a translation promoting effect is located upstream of the polynucleotide encoding GFP or mRuby, ie, the untranslated region. No mutation has occurred in the expressed GFP or mRuby.

[Example 2] Fluorescence intensity of defective mlcR-GFP transformant Instead of the full-length nucleotide sequence of mlcR, each plasmid vector was replaced with a partial nucleotide sequence in which a predetermined region was deleted from the 5 'end of mlcR. Escherichia coli K-12-derived strain HST08 was transformed and cultured in the same manner as in Example 1, and the results of observation of the fluorescence intensity of GFP with a fluorescence microscope are shown in FIG. In FIG. 6, (a) is a diagram showing the site of deletion of mlcR, and (b) is a diagram showing the GFP fluorescence intensity (Z score) of the strain transformed with each plasmid vector. Further, in FIG. 6, “mlcR_10”, “mlcR_20”, “mlcR_25”, “mlcR_30”, “mlcR_40”, “mlcR_45”, “mlcR_60”, “mlcR_74”, “mlcR_110”, “mlcR_235”, “mlcR_265”, “mlcR_265”, “mlcR_355”, and “ulc” are respectively ulC_lC. The prepared plasmid vectors "mlcR_10-RBS-Spacer-GFP-pUC19", "mlcR_20-RBS-Spacer-GFP-pUC19", "mlcR_25-RBS-Spacer-GFP-pUC19", "mlcR_30-RBS-Spacer-GFP-pUC19" mlcR_40-RBS-Spacer-GFP-pUC19 "" mlcR_45-RBS-Spacer-GFP-pUC19 " mlcR_60-RBS-Spacer-GFP-pUC19, mlcR_74-RBS-Spacer-GFP-pUC19, mlcR_110-RBS-Spacer-GFP-pUC19, mlcR_235-RBS-Spacer-GFP-pUC19, The strains transformed with "GFP-pUC19", "mlcR_355-RBS-Spacer-GFP-pUC19" and "mlcR-RBS-Spacer-GFP-pUC19" are shown. Further, GFP represents a strain transformed with the plasmid vector GFP / pUC19, and RBS + Spacer represents a strain transformed with the plasmid vector "RBS-Spacer-pUC19".

  As shown in FIG. 6 (b), GFP fluorescence was observed even when a part of the 5c end of the mlcR gene was deleted, and it was confirmed that GFP was expressed. In particular, with respect to the fluorescence intensity, when a partial base sequence of 10 bases from the 3 'end of mlcR was used, the Z score was 27 times as large as that of the control RBS + Spacer. Further, when a partial sequence of 40 bases, 235 bases, and 265 bases from the 3 ′ end of the mlcR gene is used, it has the same Z score as when the full length of the mlcR gene is used regardless of the partial base sequence, and the mlcR gene When the partial base sequence of 25 bases from the 3 'end of was used, the Z score was about twice as large as that when the full length of the mlcR gene was used. Further, as shown in FIG. 6 (b), almost no fluorescence was observed in “RBS + Spacer” and “GFP”. From these results, the polynucleotide containing at least the 474th to 483rd nucleotides of the nucleotide sequence encoding the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1 and the polynucleotide encoding the ribosome binding site, It became clear that the downstream gene can be highly expressed.

Example 3 Effect of GFP Expression on Growth and Yield of Plasmid Vector It was examined whether there was any adverse effect on the growth of host cells by expressing GFP. First, the growth rates of bacteria with and without GFP expression were compared. Culture was performed in LB medium. As a result, the growth of a strain without GFP fluorescence (pUC19 strain: a strain transformed with the plasmid vector “pUC19”) and that of a strain with GFP fluorescence (mlcR-GFP / pUC19 strain) were comparable (FIG. 7A). ). In FIG. 7A, the horizontal axis represents the culture time, and the vertical axis represents the results of absorbance measurement of relative cell density when the culture start time was set to 1. Further, a plasmid vector was purified from each strain by a known method, and the yield of the plasmid vector was compared. The yield of the plasmid vector was determined by measuring the absorbance at 260 nm using a spectrophotometer (Shimadzu Corporation). As a result, the yield of the strain with GFP fluorescence (mlcR-GFP / pUC19 strain) was about twice as high as that of the pUC19 strain without GFP fluorescence (FIG. 7B). From the above results, it was clarified that no adverse effect on the growth due to GFP expression was observed, and that the yield of the plasmid vector was large.

[Example 4] Control of expression level by glucose When expression is performed using Escherichia coli, it may be necessary to control the expression level, that is, to increase the expression level or to suppress the expression level. Since the pUC19 vector used in the above Examples has CAP, lac promoter, and lac operator as shown in FIG. 2, it was examined whether or not the expression could be controlled (catabolite repression) by glucose.

  Four types of plasmid vectors “mlcR-RBS-Spacer-GFP-pUC19”, “mlcR_25-RBS-Spacer-GFP-pUC19”, “mlcR_10-RBS-Spacer-GFP-pUC19”, and “RBS” prepared in the above Examples. -Spacer-pUC19 "was introduced into a chemically competent E. coli BL21 (DE3) strain (Cosmo Bio Inc.) prepared by a known method, and the LB medium containing ampicillin and 1% glucose, or the LB medium containing ampicillin and glucose free. Cultured overnight. The GFP fluorescence Z score of the culture solution after the culturing was determined using an inverted fluorescence microscope in the same manner as described above. FIG. 8 shows the results. In FIG. 8, the black column contains glucose and the white column does not contain glucose.

  As shown in FIG. 8, in the absence of glucose, it was revealed that GFP was highly expressed using the present vector containing the mlcR gene. Specifically, as compared with the case where only RBS is used as the Z score, it is 3.4 times when it has mlcR_10, 8.4 times when it has mlcR_25, and when it has the full length of mlcR. 6.3 times as high. On the other hand, when glucose was added, the expression was all suppressed. Therefore, it was confirmed that protein expression can be controlled by adjusting the amount of glucose in the medium.

[Example 5] Control of expression level by IPTG Since the pUC19 vector used in the above example has a lac promoter and a lac operator as shown in Fig. 2, it was examined whether the expression could be controlled by IPTG. Was.

  The four vectors “mlcR-RBS-Spacer-GFP-pUC19”, “mlcR_25-RBS-Spacer-GFP-pUC19”, “mlcR_10-RBS-Spacer-GFP-pUC19”, and “RBS-” "Spacer-pUC19" was introduced into Escherichia coli BL21 (DE3) strain (manufactured by Cosmo Bio) and cultured overnight in an LB medium containing ampicillin and 1% glucose. A part of the culture solution was further cultured in an LB medium containing ampicillin and 1 mM IPTG and containing no glucose, or an LB medium containing ampicillin and containing no glucose (without IPTG). The GFP fluorescence Z score of the culture solution after the culturing was determined using an inverted fluorescence microscope in the same manner as described above. FIG. 9 shows the results. In FIG. 9, "W /" indicates the presence of IPTG and "W / O" indicates the absence of IPTG. The horizontal axis is the culture time in the LB medium without glucose, and the vertical axis is Z score.

  As shown in FIG. 9, it was revealed that the use of the vector of the present invention containing the mlcR gene even without IPTG resulted in high expression of GFP. In addition, by adding IPTG, the expression level can be further increased by using the present vector containing the partial base sequence of the mlcR gene, particularly the partial base sequence of 25 bp from the 3 'end (not including the stop codon) in the full length 483 bp of the mlcR gene. That was confirmed. Therefore, it was confirmed that the expression could be controlled by adjusting the amount of IPTG in the medium.

  In addition, based on the results of the above Examples, 1) GFP expression can be confirmed even in a medium without IPTG using the pUC vector; 2) At this time, the transcription suppression function of LacI-LacO works in the pUC vector ( It is known that this suppression is not complete and there is "more" to be transcribed); 3) Under these circumstances, GFP which cannot be confirmed in normal times (without mlcR) due to the connection of mlcR Expression is observed; that is, "if the polynucleotide of the above (1) or (2) and the polynucleotide encoding the ribosome binding site are provided, the translation of the protein to be expressed is promoted." it is conceivable that.

  Use of the present vector enables production of useful proteins using prokaryotes.

Claims (7)

A prokaryotic expression vector comprising, in order, a polynucleotide according to the following (1) or (2), a polynucleotide encoding a ribosome binding site, and a polynucleotide encoding a protein to be expressed.
(1) The full length or partial sequence of a polynucleotide encoding the myosin regulatory light chain (mlcR) shown in SEQ ID NO: 1, which is at least the 474-483rd base of the nucleotide sequence shown in SEQ ID NO: 1. A polynucleotide comprising the sequence;
(2) a polynucleotide having at least 90% or more sequence identity with the polynucleotide of (1); The prokaryotic expression vector according to claim 1, further comprising a spacer sequence of 4 to 20 bases between the polynucleotide encoding the ribosome binding site and the polynucleotide encoding the protein to be expressed. The prokaryotic expression vector according to claim 1 or 2, wherein at least the base sequence at positions 474 to 483 of the base sequence shown in SEQ ID NO: 1 is the base sequence shown in SEQ ID NO: 2. The prokaryotic expression vector according to any one of claims 1 to 3, wherein the prokaryotic organism is Escherichia coli. A prokaryote containing the prokaryotic expression vector according to claim 1. A method for producing a protein, comprising a step of culturing the prokaryote according to claim 5 and a step of collecting a protein to be expressed from the cultured prokaryote. A kit for expressing a protein, comprising the prokaryotic expression vector according to any one of claims 1 to 4 and instructions for expressing the protein.