JP2009189306A - Protein expression vector with methionine tag - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vector simply adding a tag different from conventional histidine and cysteine to a protein, and to provide a transformant in which the vector is introduced. <P>SOLUTION: The protein expression vector with the methionine tag comprises (A) a base sequence encoding the methionine tag composed of contiguous 3-5 methionine residues, (B) a promoter sequence (wherein, the promoter sequence is connected to the upstream side of the base sequence encoding the methionine tag), and (C) a base sequence encoding a desired protein (wherein, the base sequence encoding the desired protein is connected to the downstream side of the promoter sequence and connected to the upstream side or the downstream side of the base sequence encoding the methionine tag). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein expression vector with a methionine tag, a methionine tag vector, a vector in which a base sequence encoding a protein is introduced into the methionine tag vector, and a transformant in which the vector is introduced.

  Conventionally, a method of adding a so-called tag sequence to the end of a specific protein is known in order to express the specific protein in host cells such as Escherichia coli, yeast, animal cells, etc. and to detect or purify it. . Examples of these tags (tag peptides) include a histidine tag connected with histidine and a cysteine tag connected with cysteine.

As a method for detecting a protein to which these tag peptides have been added, for example, a method using an antibody that recognizes a sequence serving as a tag is known. In addition, for example, a method for isolating a protein with a histidine tag added using zinc or cobalt has been reported (Patent Document 1). In addition, for example, a method has been reported in which a protein with a histidine tag added is obtained by using a nickel affinity column (Patent Document 2).
Special Table 2007-505134 Japanese Patent Laid-Open No. 10-212298

  An object of the present invention is to provide a vector that enables a tag different from conventional histidine and cysteine to be easily added to a protein. Specifically, an object of the present invention is to provide a vector that allows a methionine tag to be easily added to a protein. Another object of the present invention is to provide a transformant introduced with the vector.

The present inventors have studied the above problems and succeeded in constructing a vector for adding a methionine tag to a protein. The present inventors have succeeded in constructing a vector having a base sequence encoding a methionine residue and a base sequence encoding a desired protein. Furthermore, the present inventors have found that a transformant can be produced by introducing this vector into a host cell, and a desired protein to which a methionine tag has been added can be expressed in the transformant. The present invention has been completed as a result of further studies based on this finding, and is described below.
Item 1. A methionine-tagged protein expression vector comprising the following (A) to (C).
(A) a base sequence encoding a methionine tag consisting of 3 to 5 consecutive methionine residues,
(B) a promoter sequence (wherein the promoter sequence is connected upstream of the base sequence encoding the methionine tag), and
(C) a base sequence encoding a desired protein (where the base sequence encoding the desired protein is connected downstream of the promoter sequence and upstream or downstream of the base sequence encoding the methionine tag) It is connected to the.)
Item 2. A methionine tag vector containing the following (D) to (F)
(D) a base sequence encoding a methionine tag consisting of 3 to 5 consecutive methionine residues,
(E) a promoter sequence (wherein the promoter sequence is connected upstream of the base sequence encoding the methionine tag), and
(F) a cloning site for introducing a base sequence encoding the desired protein (wherein the cloning site is connected downstream of the promoter sequence and upstream of the base sequence encoding the methionine tag) (Or connected downstream).
Item 3. A protein expression vector with a methionine tag, wherein a base sequence encoding a desired protein is introduced into a cloning site connected to the vector according to Item 2.
Item 4. Item 4. A transformant obtained by introducing the vector according to Item 1 or 3 into a host cell and transforming it.

  According to the methionine-tagged protein expression vector of the present invention, in a transformant obtained by introducing the methionine-tagged protein into a host cell, a desired methionine tag consisting of consecutive 3 to 5 methionine residues is added. Protein can be expressed easily.

  Further, according to the methionine tag vector of the present invention, desired protein expression in which a methionine tag consisting of 3 to 5 consecutive methionine residues is added by inserting a base sequence encoding the desired protein into the vector. Vectors can be easily produced. In addition, by introducing the vector thus prepared into a host cell, a desired protein with a methionine tag consisting of 3 to 5 consecutive methionine residues can be easily obtained in the resulting transformant. Can be expressed.

  Further, by using these vectors, a desired protein to which a methionine tag consisting of 3 to 5 consecutive methionine residues is added can be obtained.

The present invention will be described below.
(1) Methionine-tagged protein expression vector The methionine-tagged protein expression vector of the present invention (hereinafter sometimes referred to as an expression vector) comprises (A) a base sequence encoding a methionine tag, (B) a promoter sequence, and ( C) At least a base sequence encoding a desired protein.

  The base sequence encoding the methionine tag consists of a base sequence encoding 3-5 consecutive methionine residues. When the vector used for constructing the expression vector (hereinafter also referred to as a basic vector) includes a base sequence encoding a methionine residue in advance, this may be a part of the methionine tag or It can be used as a whole. The base sequence encoding the methionine residue previously contained in the basic vector includes a base sequence corresponding to the starting methionine. That is, the base sequence encoding the methionine tag contained in the expression vector is (i) obtained by introducing a base sequence encoding 3 to 5 consecutive methionine residues into the basic vector. Or (ii) a base sequence encoding 3 to 5 consecutive methionine residues (which may be the starting methionine) previously contained in the basic vector, and (iii) It may be a base sequence encoding 3 to 5 consecutive methionine residues obtained by combining a base sequence introduced into the basic vector and a base sequence previously contained in the basic vector. The base sequence encoding the methionine tag preferably consists of a base sequence encoding 3 to 4 consecutive methionine residues.

  Therefore, the methionine tag expressed by using the expression vector is expressed only from the base sequence introduced into the basic vector, or expressed only from the base sequence previously contained in the basic vector. Alternatively, it is expressed from a base sequence obtained by combining a base sequence introduced into the basic vector and a base sequence previously contained in the basic vector.

  The methionine tag in the present invention consists of 3 to 5 consecutive methionine residues, preferably 3 to 4 consecutive methionine residues.

  In the transformant obtained by introducing the expression vector, a desired protein to which a methionine tag consisting of 3 to 5 consecutive methionine residues is added is expressed at the connection position of the promoter sequence in the expression vector. As long as it is not limited. For example, the promoter sequence is connected upstream of the base sequence encoding the methionine tag. In the expression vector, the base sequence encoding the methionine tag is under the control of the promoter sequence.

  The promoter used here will not be limited if it is a conventionally well-known promoter in the said field | area. For example, T7lac, T7, araBAD promoters and the like can be mentioned as promoters, and these are used when, for example, proteins are expressed in E. coli. Moreover, SV40, CMV, RSV, TK, EF-1α promoter and the like can be mentioned, and these are used, for example, for expressing proteins in animal cells. Other promoters that can be used include yeast promoters, insect cell promoters, and viral promoters.

  In addition, the connecting position of the base sequence encoding the desired protein in the expression vector is also added with a methionine tag consisting of 3 to 5 consecutive methionine residues in the transformant obtained by introducing the expression vector. There is no limitation as long as the desired protein is expressed.

  For example, the base sequence encoding the desired protein is connected downstream from the promoter sequence, and is connected upstream or downstream of the base sequence encoding the methionine tag. Whether the base sequence encoding the desired protein is connected upstream or downstream of the base sequence encoding the methionine tag depends on the nature of the desired protein used, the nature of the host cell, etc. The change is made in accordance with the above, and the change is made within a range generally considered in the field.

  For example, when the base sequence encoding the desired protein is connected downstream of the base sequence encoding the methionine tag, and there is a signal sequence such as methionine at the N-terminal part of the base sequence encoding the desired protein, This may be cleaved by the action of an enzyme such as methionine protease at the N-terminal part. In such a case, preferably, the base sequence encoding the desired protein is connected downstream from the promoter sequence, and is connected upstream from the base sequence encoding the methionine tag.

  In any case, in the expression vector, the base sequence encoding the desired protein is under the control of the promoter sequence.

  The type of the desired protein is not limited, and any protein can be used.

  In addition, the expression vector is connected with other factors such as a replication origin and a ribosome binding site generally required for protein expression, and also includes a green fluorescent protein (GFP) gene, a luciferase gene, a chloramphenicol acetyltransferase. A base sequence such as a (CAT) gene, β-glucuronidase gene, drug resistance gene, or a base sequence encoding a β-galactosidase (LacZ gene) may be connected. These base sequences are connected to any position of the expression vector as long as they do not prevent suitable expression of the methionine tag and the desired protein.

  As a method for producing the expression vector, a method known in the art can be used, and it is not limited as long as a desired expression vector can be obtained.

  For example, the introduction of a base sequence encoding the desired protein into a basic vector may be performed by placing a cloning site together with the base sequence encoding the methionine tag on the basic vector and using a restriction enzyme or the like at the cloning site. It may be carried out by introducing a base sequence encoding the protein.

  In addition, for example, the introduction of a base sequence encoding the methionine tag into a basic vector is performed by placing a cloning site together with a base sequence encoding a desired protein on the basic vector, and adding a restriction enzyme or the like to the cloning site portion. May be used by introducing a base sequence encoding a methionine tag.

  In addition, for example, the introduction of the base sequence encoding the methionine tag and the base sequence encoding the desired protein into the basic vector is performed by previously combining the base sequence encoding the methionine tag and the base sequence encoding the desired protein. This may be done by connecting and introducing it into the basic vector.

As the vector (basic vector) used for constructing the expression vector, various types can be used and are not limited as long as a desired protein expression vector can be obtained. Examples of the vector include vectors that do not contain a promoter such as pUC18 / 19, pBR322, and pBluescript, and vectors that already contain a promoter such as pET vector, pCruz expression vector, and pcDNA vector. These vectors may be appropriately selected depending on the intended use within the range generally considered in the field.
(2) Methionine tag vector The methionine tag vector of the present invention (hereinafter sometimes referred to as tag vector) comprises (D) a base sequence encoding a methionine tag, (E) a promoter sequence, and (F) a cloning site at least. Have.

  The base sequence encoding the methionine tag is composed of a base sequence encoding 3 to 5 consecutive methionine residues, and the same base sequence as that encoding the (A) methionine tag is used.

  That is, when the vector used for constructing the tag vector of the present invention (hereinafter also referred to as a basic vector) contains a sequence encoding a methionine residue (may be the starting methionine) in advance. Can be used as part or all of the methionine tag. Therefore, the base sequence encoding the methionine tag contained in the tag vector can also be obtained by introducing (i) a base sequence encoding 3-5 consecutive methionine residues into the basic vector. Or (ii) a base sequence encoding 3 to 5 consecutive methionine residues previously contained in the basic vector, or (iii) a base introduced into the basic vector. It may be a base sequence encoding 3 to 5 consecutive methionine residues obtained by combining the sequence and a base sequence previously contained in the basic vector.

  The base sequence encoding the methionine tag preferably consists of a base sequence encoding 3 to 4 consecutive methionine residues.

  Therefore, the methionine tag expressed using the tag vector is expressed only from the base sequence introduced into the basic vector, or is expressed only from the base sequence previously contained in the basic vector. Alternatively, it is expressed from a base sequence obtained by combining a base sequence introduced into the basic vector and a base sequence previously contained in the basic vector.

  The methionine tag in the present invention also consists of 3 to 5 methionine residues that are continuous, and preferably 3 to 4 methionine residues that are continuous.

  The connection position of the promoter sequence in the tag vector is not limited as long as a vector having a desired effect is obtained. For example, the promoter sequence is connected upstream of the base sequence encoding the methionine tag. In the tag vector, the base sequence encoding the methionine tag is under the control of the promoter sequence. The promoter used here is not limited as long as it is a promoter known in the art, and examples thereof are those described above.

  In addition, the connection position of the cloning site in the tag vector is not limited as long as a vector having a desired effect is obtained. For example, the cloning site is connected downstream of the promoter sequence and upstream and / or downstream of the base sequence encoding the methionine tag.

  Whether the cloning site is connected upstream or downstream of the base sequence encoding the methionine tag, the nature of the protein that can be introduced into the cloning site, and further the host cell The change is made as appropriate according to the properties and the like, and the change is made within a range generally considered in the field.

  For example, the cloning site is connected downstream of the base sequence encoding the methionine tag, and there is a signal sequence such as methionine at the N-terminal part of the base sequence encoding a protein that can be subsequently introduced into the cloning site. In some cases, this may be cleaved by the action of an enzyme such as methionine protease at the N-terminal part. In such a case, the cloning site is preferably connected upstream of the base sequence encoding the methionine tag.

  When the cloning site is connected upstream and downstream of the base sequence encoding the methionine tag, the base sequence encoding the protein that can be introduced thereafter is the cloning of either the upstream part or the downstream part. It may be introduced to the site. In this case, the cloning site into which the nucleotide sequence encoding the protein is to be introduced is appropriately determined according to the properties of the protein that can be introduced into the cloning site as described above, and further the properties of the host cell. The

  The cloning site is connected to introduce a base sequence encoding a desired protein into the tag vector, and this is not limited, and examples include a multiple cloning site. For this reason, the cloning sites are connected such that the base sequence encoding the desired protein introduced in the tag vector is under the control of the promoter sequence.

  The kind of the desired protein is not limited, and any protein can be used.

  In addition, the tag vector is connected to a factor generally required for protein expression, and may be connected to a base sequence encoding a gene such as a GFP gene, a luciferase gene, or a drug resistance gene. The base sequence and the introduction site are exemplified above.

  The arrangement of a base sequence encoding a methionine tag, a promoter sequence, and a cloning site in the tag vector is illustrated in FIG. In the tag vector shown in FIG. 1, a multicloning site (MCS) is connected upstream and downstream of a base sequence (tag) encoding a methionine tag, and a promoter sequence (promoter) is further upstream of the upstream multicloning site. ) Is connected. In addition, in the tagged vector shown in FIG. 1, the origin of replication (Ori) and drug resistance gene are also shown.

The method for producing the tag vector is not limited as long as a method known in the art can be used and a desired tag vector can be obtained. Moreover, the above-mentioned thing is illustrated as a vector (basic vector) used in order to construct | assemble the said tag vector.
(3) Introduction of a base sequence encoding a protein into a tag vector A base sequence encoding a desired protein can be introduced into the tag vector. Preferably, the base sequence encoding the desired protein is introduced upstream or downstream of the base sequence encoding the methionine tag. More preferably, the base sequence encoding the desired protein is introduced into a cloning site connected to the tag vector. Here, the desired protein is not limited.

  Introduction of the base sequence encoding the desired protein into the tag vector is performed according to a method known in the art. For example, when a base sequence encoding the desired protein is introduced into a cloning site, the base sequence can be introduced at a target position by using a restriction enzyme recognition site contained in the cloning site. .

Thus, by introducing a base sequence encoding a desired protein into the tag vector, a base sequence consisting of 3 to 5 methionine residues continuous on the N-terminal side or C-terminal side of the base sequence is added. be able to.
(4) Transformant The transformant of the present invention is introduced into a host cell by introducing the expression vector into a host cell or after introducing a base sequence encoding a desired protein into the tag vector. Can be obtained.

  Here, the host cell is not limited, and examples thereof include various cells such as bacteria such as E. coli, yeast, insect cells, plant cells, and animal cells. Bacteria and animal cells are preferred.

  In addition, introduction of the expression vector or a tag vector into which a base sequence encoding a desired protein has been introduced into a host cell can be performed according to a conventionally known method in the art.

  For example, these vectors can be introduced into Escherichia coli by introducing the heat shock method into a competent cell prepared by the calcium chloride method or rubidium chloride method, or by electroporation by introducing a hole in the cell membrane. The method etc. can be used. Further, for example, Li method for yeast cells, Agrobacterium method, particle gun method, electroporation method for plant cells, and calcium chloride method, liposome method, DEDE dextran method, lipofection method, electroporation method for mammalian cells. For example, an injection method, a microinjection method, a particle gun method, and a virus method.

  By using the transformant thus obtained, in the transformant, a protein to which a methionine tag consisting of 3 to 5 methionine residues continuous at the N-terminus or C-terminus is added is expressed. Can do.

  Expression of the protein in such a transformant can be performed according to a conventionally known method in the art. For example, as described in the following Examples, the protein to which the methionine tag is added can be expressed through a step of culturing the transformant in a suitable medium.

  In addition, the protein thus expressed can be recovered and purified according to a conventionally known method in the art. For example, these proteins can be recovered and purified according to the methods described in the following examples. These proteins can be recovered and purified using a methionine tag consisting of 3 to 5 consecutive methionine residues and gold. Examples of the method for recovering using gold include the methods described in the following examples.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to these Examples.
Example 1
The methionine-added expression vector of the present invention was constructed using a vector pET-15b (MERCK / Novagen) that allows protein expression in E. coli. As the desired protein, Enhanced Green Fluorescent Protein (EGFP) often used as a marker protein was used. Using pCruz GFP Expression vector C (Santa Cruz Biotechnology, Inc.) containing the above-mentioned protein EGFP, between the ScaI and BglII sites of the multiple cloning site (MCS) existing on the 3 'end side of the base sequence encoding EGFP. Annealed oligonucleotides (5′-CATGATGATGTA-3 ′ and 5′-GATCTACATCATCATG-3 ′) having a methionine tag introduction sequence containing a base sequence encoding the following three methionines were introduced. The former primer is primer 1-1 and is represented by SEQ ID NO: 1. The latter primer is designated as primer 1-2 and is represented by SEQ ID NO: 2.

  Specifically, 5 μg of pCruz GFP Expression vector C was treated with restriction enzyme ScaI (Toyobo Co., Ltd.) 25 U for 16 hours at 37 ° C. in 100 μl using the attached buffer conditions, Use complete cleavage was confirmed by electrophoresis. Furthermore, restriction enzyme BglII (manufactured by Toyobo Co., Ltd.) 25 U was added to the obtained treatment solution, followed by treatment at 37 ° C. for 3 hours, and then complete cleavage was confirmed by electrophoresis using a part of the treatment solution. After confirmation, the obtained treatment solution was diluted to 200 μl with TE (purified water containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA), and the volume ratio was phenol: chloroform: isoamyl alcohol = 25. : 200 μl of a 24: 1 mixture was added and mixed with a vortex mixer to inactivate the enzyme. Thereafter, centrifugation was performed using a centrifuge M150-IV (manufactured by Sakuma Seisakusho Co., Ltd.) at 15000 rpm for 5 minutes at room temperature, and the supernatant was collected. To the supernatant, add 1 μl of 10 μg / μl glycogen aqueous solution, 20 μl of 3M sodium acetate aqueous solution (pH 5.2), and 500 μl of ethanol (special grade: manufactured by Nacalai Tesque). Mix well, then at 15000 rpm for 5 minutes at 4 ° C. Centrifugation was performed and the supernatant was removed. Further, 500 μl of a 70% by volume ethanol aqueous solution was added thereto, suspended, and then centrifuged at 15000 rpm for 5 minutes at 4 ° C., and the supernatant was removed and vacuum dried. This was dissolved in 30 μl of TE, and a portion thereof was subjected to electrophoresis to confirm whether the cleaved pCruz GFP Expression vector C was recovered, and the concentration was estimated using a molecular weight marker.

  On the other hand, each oligonucleotide containing a methionine tag introduction sequence was dissolved in TE to a concentration of 100 pmol / μl, and 5 μl each and 25 μl of 5M NaCl aqueous solution were made up to 500 μl with TE and heat-treated at 95 ° C. for 5 minutes. After that, it was gradually cooled for about one and a half hours (the solution concentration after annealing was 10 pmol / μl). Using 150 fmol or 300 fmol, this was introduced into pCruz GFP Expression vector C 50 fmol, which had been previously treated with the restriction enzyme, at 16 ° C. for 30 minutes using a ligation kit (Takara Bio Inc.). After the introduction, the resulting solution was made up to 200 μl with TE, and 200 μl of a mixed solution of phenol: chloroform: isoamyl alcohol = 25: 24: 1 was added and mixed with a vortex mixer to inactivate the enzyme. . Further, centrifugation was performed at 15000 rpm for 5 minutes at room temperature, and the supernatant was separated. To the supernatant, 1 μl of 10 μg / μl glycogen aqueous solution, 20 μl of 3M sodium acetate aqueous solution and 500 μl of ethanol were added and mixed well, followed by centrifugation at 15000 rpm for 5 minutes at 4 ° C. to remove the supernatant. Further, 500 μl of 70 vol% ethanol aqueous solution was added and suspended, followed by centrifugation at 15000 rpm for 5 minutes at 4 ° C., and the supernatant was removed and vacuum dried. This was dissolved in 5 μl of TE, and then treated with restriction enzyme ScaI 5 U at 37 ° C. for 30 minutes in 50 μl under the attached buffer conditions. Further, the volume was increased to 200 μl with TE, 200 μl of a mixed solution with a volume ratio of phenol: chloroform: isoamyl alcohol = 25: 24: 1 was added and mixed with a vortex mixer to deactivate the enzyme. Centrifugation was performed at 15000 rpm for 5 minutes at room temperature, and the supernatant was collected. To the supernatant, 20 μl of 3M aqueous sodium acetate solution and 500 μl of ethanol (Nacalai Tesque) were added and mixed well, followed by centrifugation at 15000 rpm, 5 min, 4 ° C. to remove the supernatant. Further, 500 μl of 70% by volume ethanol aqueous solution was added and suspended, followed by centrifugation at 15000 rpm for 5 minutes at 4 ° C., the supernatant was removed, vacuum dried, and then dissolved in 5 μl of TE. Add 50 μl of competent cell DH5α to the total volume of this solution, leave it on ice for 30 minutes, leave it at 42 ° C. for 60 seconds, and leave it on ice for 3 minutes, add 450 μl of SOC medium, and incubate at 37 ° C. for 1 hour. Amp +) plate. After 16 hours at 37 ° C., a plasmid was prepared from the obtained colonies, and those containing the introduced methionine tag were identified by confirming the nucleotide sequence of the plasmid.

  PCR was performed using the obtained plasmid as a template and primers (5'-TAATACGACTCACTATAGGG-3 ', 5'-TAGAAGGCACAGTCGAGG-3'). The former primer is primer 2-1 and is represented by SEQ ID NO: 3. The latter primer is designated as primer 2-2 and is represented by SEQ ID NO: 4. The EGFP part containing the amplified methionine tag is treated with restriction enzymes NcoI and BglII, and the resulting fragment (sequence with the base sequence encoding the methionine tag introduced on the C-terminal side of EGFP) is the NcoI and BamHI sites of pET-15b. And an EGFP expression vector with methionine tag for E. coli was prepared.

  Specifically, PCR reaction was carried out according to the protocol attached to EX Taq (Takara Bio Inc.) using 0.5 μl of the resulting plasmid diluted 100-fold as a template and 2.5 fmol of each of the primers described above. I did it. The PCR cycle was 95 ° C., 30 seconds, (95 ° C., 10 seconds, 55 ° C., 20 seconds, 72 ° C., 60 seconds) × 30 cycles, 72 ° C., 4 minutes, held at 15 ° C. After the reaction, the obtained PCR product was purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega) according to the attached protocol. The total amount of the obtained solution was treated with 20 U each of restriction enzymes NcoI (NEB) and BclII (NEB) in 100 μl at 37 ° C. for 2 hours using the attached buffer conditions. The entire amount was electrophoresed in 7% SEAKEM® GTG® Agarose (CAMBREX) gel in TAB buffer at 100 V constant pressure for 30 minutes. The confirmed migration band at a predetermined position was cut out and purified from the gel using Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega) according to the attached protocol. The obtained fragment solution was partially electrophoresed, and the concentration was estimated from the size marker.

  Plasmid pET-15b to be introduced was treated with 5 μg of restriction enzyme NcoI (Takara Bio Inc.), BamHI (Takara Bio Inc.) 25 U each in 100 μl using the attached buffer conditions at 37 ° C. for 16 hours. . After confirming by 7% by weight agarose (Takara Bio Inc.) gel electrophoresis using a portion, make up to 200 μl with TE (purified water containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA) 200 μl of a mixture of phenol: chloroform: isoamyl alcohol = 25: 24: 1 was added and mixed with a vortex mixer to deactivate the enzyme. Centrifugation was performed at 15000 rpm for 5 minutes at room temperature, and the supernatant was collected. To the supernatant, 1 μl of a 10 μg / μl glycogen aqueous solution, 20 μl of 3 M sodium acetate aqueous solution and 500 μl of ethanol were added and mixed well, followed by centrifugation at 15000 rpm for 5 minutes at 4 ° C. to remove the supernatant. Further, 500 μl of 70 vol% ethanol aqueous solution was added, suspended, centrifuged at 15000 rpm for 5 minutes at 4 ° C., and the supernatant was removed and vacuum dried. This was dissolved in 20 μl of TE, treated with Alkaline phosphatase (BAPC75, Takara Bio Inc.) at 65 ° C. for 30 minutes according to the attached protocol, and further added with 1 μl of enzyme and further treated at 65 ° C. for 30 minutes. After the reaction, the volume was increased to 200 μl with TE, and 200 μl of a mixed solution of phenol: chloroform: isoamyl alcohol = 25: 24: 1 was added and mixed with a vortex mixer to deactivate the enzyme. The obtained solution was centrifuged at 15000 rpm for 5 minutes at room temperature, and the lower layer phenol was separated, and again, 200 μl of a mixed solution with a volume ratio of phenol: chloroform: isoamyl alcohol = 25: 24: 1 In addition, the enzyme was completely inactivated by mixing with a vortex mixer. The supernatant was separated, 20 μl of 3M sodium acetate aqueous solution and 500 μl of ethanol were added and mixed well, followed by centrifugation at 15000 rpm for 5 minutes at 4 ° C. to remove the supernatant. Further, 500 μl of 70% by volume ethanol aqueous solution was added and suspended, followed by centrifugation at 15000 rpm for 5 minutes at 4 ° C., the supernatant was removed, vacuum dried, and then dissolved in 30 μl TE. A portion was electrophoresed to confirm whether it was recovered, and the concentration was estimated using a molecular weight marker.

  This plasmid 50 fmol and the previously recovered fragment solution 50 fmol were reacted at 16 ° C. for 30 minutes according to the protocol attached to the ligation kit (Takara Bio Inc.), and a part thereof was transformed into competent cell DH5α. A plasmid was prepared from the obtained colonies, and a plasmid having a methionine tag on the C-terminal side of EGFP in pET-15b was obtained. In order to confirm whether the base sequence encoding the methionine tag exists on the C-terminal side of the base sequence encoding EGFP, the entire base sequence of the introduced sequence was confirmed.

Similarly, by using the following sequences (5′-CATGATGATGATGATGTA -3 ′ and 5′-GATCTACATCATCATCATCATG -3 ′) containing the sequences encoding five methionines in the methionine tag introduction primer shown above, methionine is 5 An individual methionine-tagged EGFP expression vector was obtained. The former primer is primer 3-1 and is represented by SEQ ID NO: 5. The latter primer is designated as primer 3-2 and represented by SEQ ID NO: 6.
Example 2
Methionine-tagged proteins can be expressed and recovered by known methods. In Example 2, experiments were conducted using recombinant E. coli obtained by transforming the methionine-tagged EGFP expression vector prepared in Example 1 into E. coli. The following operations were performed for the methionine-tagged EGFP expression vector with three methionine and the methionine-tagged EGFP expression vector with five methionine.

  Specifically, the transformation was performed as follows. About 10 ng of the methionine-tagged EGFP expression vector plasmid prepared in Example 1 is added to 100 μl of competent cell BL21 (DE3), left on ice for 30 minutes, heat-treated at 42 ° C. for 60 seconds, and quickly returned to ice. Let stand for a minute. Then, 1 ml of SOC medium was added, incubated at 37 ° C., applied to an LB (carbenicillin added) plate, and incubated at 37 ° C. for 16 hours to obtain a colony obtained as a transformant.

The resulting transformant is cultured at 30 ° C in an LB liquid medium containing 50 µg / ml carbenicillin, and expression is induced by adding IPTG to a final concentration of 1 mM in the logarithmic growth phase (around OD ₆₀₀ = 0.6). 3 hours later, E. coli was recovered. From this, all the following operations were performed at 4 ° C.

The recovered E. coli was washed twice with Tris buffer (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM DTT), suspended in Tris buffer containing protease inhibitor (Nacalai Tesque), and sonicated on ice. The resulting suspension is centrifuged, and the supernatant is filtered through a 0.45 μm filter and purified using the Econo gradient pump system (Bio-Rad) and the Econo pack ion exchange cartridge High Q (Bio-Rad). did. At this time, EGFP was bound to the column using Tris buffer, and proteins other than EGFP were removed by raising the NaCl concentration to 80 mM. Then, apply a concentration gradient from 80 mM to 110 mM, collect the EGFP-containing fraction at around 110 mM, and concentrate to purify EGFP (without methionine tag). Similarly, purify EGFP protein with methionine tag. I did it.
Example 3
A methionine-tagged protein is specifically and efficiently separated by a method using gold-attached magnetic metal oxide particles (provided by Professor Takao Yamamoto, Faculty of Engineering, Osaka University; hereinafter referred to as gold magnetic particles). An attempt was made to purify.

Here, γ-Fe ₃ O ₄ was used as the magnetic metal oxide particles, and the gold magnetic particles were produced according to the method described in WO 2004/083124. In addition, this experiment was performed for EGFP with three methionines added as the methionine tag, EGFP with five methionines added, and EGFP without the methionine tag obtained in Example 2.

  Each methionine-tagged EGFP protein or untagged EGFP protein prepared in Example 2 was replaced with a buffer (10 mM phosphate buffer) while being concentrated with an ultrafiltration system. Further, the gold magnetic particles were previously washed with the same buffer. Each EGFP protein solution was measured for fluorescence intensity (no unit), and then diluted with a buffer so as to be 200 (fluorescence intensity) / μl. 100 μl of these EGFP protein solutions were added to 400 μl of gold magnetic particles (Fe 5 mg / ml) and mixed with a rotary shaker (4 ° C., 60 minutes) to bind the gold magnetic particles and the EGFP protein. Thereafter, the gold magnetic particles bound with the EGFP protein were allowed to stand for 30 minutes on a magnet (magnetic separation), and the supernatant was collected. The supernatant was subjected to fluorescence measurement, and the amount of EGFP protein bound to the gold magnetic particles was determined by subtracting the amount of fluorescence of the solution before binding from the obtained value.

  Furthermore, the residue containing the conjugate of the gold magnetic particles and the EGFP protein recovered by the magnetic separation is suspended in a buffer, and after the magnetic separation, the supernatant is removed twice to obtain the gold magnetic particles. After removing non-specific adsorbate and the like, the suspension was suspended in 500 μl of a buffer containing 12 mM mercaptoethanol, and then EGFP protein was eluted while mixing on a rotary shaker at 4 ° C. for 30 minutes. Then, the amount of fluorescence after magnetic separation was measured, and this was used as the amount of EGFP protein eluted from the above conjugate. The results are shown in FIG.

  Fluorescence is measured in a 96-well plate with 100 μl of measurement sample, measured using a fluorescence plate reader Cyto Fluor II (PerSeptive Biosystems) (use filters are Ex; 485/20, Em: 530/25), and the background is subtracted. The amount of binding and the amount of elution were determined in terms of the actual liquid volume.

  As a result, the amount of binding increased when the tag region had 3 and 5 methionines compared to those without the tag region. In addition, the amount of elution was increased with the methionine tag compared to the case without the tag. A significant difference was observed in both the amount of binding and the amount of elution compared to the case where there was no methionine in the tag region (**: P <0.01, *; p <0.05).

An example of the arrangement of a base sequence encoding a methionine tag, a promoter sequence, and a cloning site in a tag vector is shown. The site of the methionine tag is expressed as tag. There is a promoter sequence upstream of the tag site, and there is an MCS for introducing a base sequence encoding the desired protein between the promoter sequence and the tag site and immediately downstream of the tag site. This vector also contains a drug resistance gene necessary for production of a transformant and an origin of replication (ori) necessary for replication of the vector in E. coli. The result of Example 3 is shown. Using three types with different particle creation dates, the amount of binding and elution of each purified tag protein was determined, and the average and standard deviation were taken and shown in the graph.

  SEQ ID NO: 1 shows the base sequence of primer 1-1.

  SEQ ID NO: 2 shows the base sequence of primer 1-2.

  SEQ ID NO: 3 shows the base sequence of primer 2-1.

  SEQ ID NO: 4 shows the base sequence of primer 2-2.

  SEQ ID NO: 5 shows the base sequence of primer 3-1.

  SEQ ID NO: 6 shows the base sequence of primer 3-2.

Claims (4)

A methionine-tagged protein expression vector comprising the following (A) to (C).
(A) a base sequence encoding a methionine tag consisting of 3 to 5 consecutive methionine residues,
(B) a promoter sequence (wherein the promoter sequence is connected upstream of the base sequence encoding the methionine tag), and
(C) a base sequence encoding a desired protein (where the base sequence encoding the desired protein is connected downstream of the promoter sequence and upstream or downstream of the base sequence encoding the methionine tag) It is connected to the.) A methionine tag vector comprising the following (D) to (F).
(D) a base sequence encoding a methionine tag consisting of 3 to 5 consecutive methionine residues,
(E) a promoter sequence (wherein the promoter sequence is connected upstream of the base sequence encoding the methionine tag), and
(F) a cloning site for introducing a base sequence encoding the desired protein (wherein the cloning site is connected downstream of the promoter sequence and upstream of the base sequence encoding the methionine tag) (Or connected downstream). A methionine-tagged protein expression vector in which a base sequence encoding a desired protein is introduced into a cloning site connected to the vector according to claim 2. A transformant obtained by introducing the vector according to claim 1 or 3 into a host cell for transformation.

Images