JP7597346B2 - Tag peptide and method for purifying polypeptide - Google Patents

Description

The present invention relates to a tag peptide, a tag peptide fusion polypeptide, a polynucleotide encoding the tag peptide, an expression vector containing the polynucleotide, a method for purifying a polypeptide, and a kit for purifying a polypeptide containing the expression vector.

When protein synthesis is performed using E. coli or yeast, genetic engineering knowledge has led to the separation and purification of the target protein or other polypeptide. A widely used protein purification method is one that uses polyhistidine and a nickel column. Specifically, this method purifies a target protein to which six consecutive histidines, i.e. a histidine tag, have been added, as shown in (1) to (4) below, using the binding ability of histidine to nickel metal.

(1) A target protein tagged with six consecutive histidines is produced in a host cell such as yeast or E. coli.
(2) A target protein extract from the host cells is passed through a nickel column to adsorb the target protein onto the nickel.
(3) A low-concentration (approximately 5 to 10 mM) imidazole solution is passed through the column to wash it. During this process, the target protein is adsorbed onto the nickel, while the rest of the protein is washed off the column.
(4) A high concentration (approximately 200 mM) imidazole aqueous solution is passed through the column to strip the target protein trapped in (2) from the nickel and elute it.

However, since many proteins containing a lot of histidine and proteins that bind to nickel metal are present within the cells of E. coli or yeast in which protein synthesis has been performed, these proteins also adsorb to the nickel column, making it difficult to purify the target protein with a high purity. In addition, when removing the target protein adsorbed to the nickel from the nickel with an elution solution, an imidazole aqueous solution is usually used, but when using the target protein, it is sometimes necessary to remove the imidazole.

Other than six consecutive histidines, tag peptides include, for example, a chelating peptide represented by the formula: (His)x-(A)y-(His)z [wherein A is one or more amino acids; x is 0-10; y is 0-4; z is 0-10] (see Patent Document 1), a peptide of the formula R1-(His)2-6-R2 (wherein R1 is hydrogen, one or several amino acids, R2 is Q, Q-Ile-Glu-Gly-Arg, or Q-Asp-Asp-Asp-Lys-, and Q is a peptide bond, one or several amino acids, up to a maximum of 30 amino acids) (see Patent Document 2), and a sequence of three or more consecutive basic amino acids (see Patent Document 3). However, most of them are eluted with imidazole, and there was no tag peptide that could be eluted with a high accuracy using a compound other than imidazole as necessary.

The inventors have also proposed a tag peptide that can bind to one or more metal ions selected from nickel, copper, zinc, and cobalt even when imidazole at a concentration of 500 mM or more is used (see Patent Document 4). Such a tag peptide is difficult to elute even with a high concentration of imidazole, making it possible to immobilize a target protein with high purity. However, it has been difficult to elute the target protein as needed.

Japanese Patent Application Publication No. 6-157600 Japanese Unexamined Patent Publication No. 63-251095 JP 2008-241560 A JP 2017-88536 A

The objective of the present invention is to provide a tag peptide that can bind to one or more metal ions selected from nickel, copper, zinc, and cobalt, and can be eluted using a substance other than imidazole.

The present inventors have been conducting research into peptide tags that can efficiently isolate and purify only the target protein, and into efficient methods for isolating and purifying proteins that have peptide tags. In the process, they confirmed that adding consecutive histidines to a protein reduces the activity of the protein. Therefore, they investigated histidine tag peptides that can suppress the decrease in protein activity.

As a result, they found that by adding a tag peptide in which aspartic acid, glutamic acid, glycine, proline, etc. are linked to histidine to the target protein, the decrease in protein activity is suppressed compared to when a tag peptide of consecutive histidines is used, and a highly active protein can be obtained. Furthermore, when they investigated elution from nickel, they found that elution is possible not only with the conventionally used imidazole, but also with low-concentration salt solutions and water, and thus completed the present invention.

That is, the present invention is as follows.
[1] A tag peptide consisting of any one of the following polypeptides (1-1) to (1-11):
(1-1) A polypeptide in which one histidine in the amino acid sequence shown in SEQ ID NO:1 is replaced with aspartic acid or glutamic acid;
(1-2) A polypeptide in which one or two histidines in the amino acid sequence shown in SEQ ID NO:2 are replaced with aspartic acid or glutamic acid;
(1-3) A polypeptide in which 1 to 3 histidines in the amino acid sequence shown in SEQ ID NO: 3 are replaced with aspartic acid or glutamic acid;
(1-4) A polypeptide in which 1 to 4 histidines in the amino acid sequence shown in SEQ ID NO: 4 are replaced with aspartic acid or glutamic acid;
(1-5) A polypeptide in which 1 to 5 histidines in the amino acid sequence shown in SEQ ID NO:5 are replaced with aspartic acid or glutamic acid;
(1-6) A polypeptide in which 1 to 6 histidines in the amino acid sequence shown in SEQ ID NO:6 are replaced with aspartic acid or glutamic acid;
(1-7) A polypeptide in which 1 to 7 histidines in the amino acid sequence shown in SEQ ID NO: 7 are replaced with aspartic acid or glutamic acid;
(1-8) A polypeptide in which 1 to 8 histidines in the amino acid sequence shown in SEQ ID NO: 8 are replaced with aspartic acid or glutamic acid;
(1-9) A polypeptide in which 1 to 9 histidines in the amino acid sequence shown in SEQ ID NO: 9 are replaced with aspartic acid or glutamic acid;
(1-10) A polypeptide in which 1 to 10 histidines in the amino acid sequence shown in SEQ ID NO: 10 are substituted with aspartic acid or glutamic acid;
(1-11) A polypeptide in which 1 to 11 histidines in the amino acid sequence shown in SEQ ID NO: 11 are replaced with aspartic acid or glutamic acid;
[2] The tag peptide according to [1] above, which comprises a tripeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 12 to 15.
[3] The tag peptide according to [1] or [2] above, which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17.
[4] A tag peptide fusion polypeptide in which the tag peptide according to any one of [1] to [3] above is fused with a polypeptide.
[5] The tag peptide fusion polypeptide according to [4] above, wherein the polypeptide fused to the tag peptide is a protein.
[6] A polynucleotide encoding the tag peptide according to any one of [1] to [3] above.
[7] An expression vector containing the polynucleotide described in [6] above.
[8] A method for purifying a polypeptide, comprising the following steps (2-1) to (2-3):
(2-1) a step of mixing a carrier having one or more metal ions selected from nickel, copper, zinc, and cobalt immobilized thereon with the tag peptide fusion polypeptide according to [4] or [5] above in the presence of an adsorption solution to adsorb the tag peptide fusion polypeptide onto the carrier;
(2-2) washing the carrier onto which the tag peptide fusion polypeptide is adsorbed with a washing solution;
(2-3) eluting the tag peptide fusion polypeptide adsorbed to the carrier from the carrier using an elution solution;
[9] The method for purifying the polypeptide according to [8] above, wherein in the step (2-1), the adsorption solution is an alkali metal salt solution having a concentration of 0.03 M or more.
[10] The method for purifying the polypeptide according to [8] or [9] above, wherein in the step (2-2), the washing solution is an alkali metal salt solution having a concentration of 0.03 M or more.
[11] The method for purifying the polypeptide according to any one of [8] to [10] above, wherein in the step (2-3), the eluent is an imidazole solution, an L-histidine solution, a buffer solution, an alkali metal salt solution or water.
[12] The method for purifying the polypeptide according to any one of [8] to [11] above, wherein the metal ion is nickel.
[13] A kit for purifying a polypeptide comprising the expression vector according to [7] above.

By using the tag peptide of the present invention, after the polypeptide is adsorbed to a carrier on which one or more metal ions selected from nickel, copper, zinc, and cobalt are immobilized, it can be eluted not only with imidazole but also with L-histidine, Tris base, or water.

FIG. 1 shows the results of evaluating the activity of tag peptide-fused red fluorescent protein (RFP) in Example 1, in which a tag peptide having various amino acids arranged between three histidines is added to the C-terminus of RFP. 1 shows the results of investigating the activity of tag peptide-fused luciferase in Example 2, in which tag peptides consisting of various combinations of histidine and aspartic acid were added to the C-terminus of Gaussia luciferase (GLuc). FIG. 13 shows the results of examining adsorption by mixing a protein extract of RFP-6HE, RFP-6HD, RFP-6H or RFP with nickel beads in Example 3. FIG. 13 shows the results of investigating adsorption by mixing a protein extract of RFP-6HE or RFP-6H with nickel beads and varying the type and concentration of the salt solution in the adsorption solution in Example 4. FIG. 13 shows the results of eluting RFP-6HE and RFP-6H adsorbed to nickel beads with imidazole in Example 5. FIG. 13 shows the results of eluting RFP-6HE and RFP-6H adsorbed to nickel beads with histidine in Example 5. FIG. 13 shows the results of eluting RFP-6HE and RFP-6H adsorbed to nickel beads with a Tris buffer solution in Example 5. 8 shows the results of eluting RFP-6HE and RFP-6H adsorbed on a nickel column with imidazole in Example 6. Figures 8(a) and (b) show the results of UV 280 nm measurement, and Figure 8(c) shows the result of SDS-PAGE analysis. FIG. 13 shows the results of eluting RFP-6HP and RFP-6HG adsorbed on a nickel column with a concentration gradient of imidazole in Example 7. FIG. 13 shows the results of eluting RFP-6HE and RFP-6H adsorbed on a nickel column without washing with distilled water in Example 7. This is a diagram showing the results of washing RFP-6HE adsorbed on a nickel column with a concentration gradient of 1 M-0.1 M and 0.1 M-0 M NaCl in Example 8. The upper row shows, from the left, photographs of the column before washing (1 M), after washing with 0.1 M NaCl, and after washing with 0.01 M NaCl, and the lower row shows the results of measuring UV 280 nm when washed with a concentration gradient of 0.1 M-0 M NaCl. In Example 8, RFP-6HE adsorbed on a nickel column was washed with a concentration gradient of 1 M-0.1 M and/or 0.1 M-0 M KCl, LiCl, or RbCl. From the left, these are photographs of the column before washing (1 M), after washing with 0.1 M, and after washing with 0.01 M. In Example 9, RFP-6HE adsorbed on a nickel column in 1 M KPO ₄ pH 8.0 was washed with 0.05 M KCl for 30 minutes, and then eluted with a concentration gradient of 0-100 mM imidazole, followed by measurement of UV at 280 nm. In Example 9, RFP-6HE adsorbed on a nickel column in 1 M KPO ₄ pH 8.0 was washed with 0.05 M KCl for 30 minutes, and then eluted with a concentration gradient of 0-100 mM L-histidine, followed by measurement of UV at 280 nm. In Example 9, RFP-6HE adsorbed on a nickel column in 1 M KPO ₄ pH 8.0 was washed with 0.05 M KCl for 30 minutes, and then eluted with distilled water for about 30 minutes, and UV at 280 nm was measured. In Example 10, DNA polymerase-6HE adsorbed on a nickel column in 1 M KPO ₄ pH 8.0 was washed with 0.05 M KCl for 30 minutes, and then eluted with 100 mM imidazole for about 30 minutes, followed by measurement of UV at 280 nm.

One aspect of the present invention is
(1-1) A polypeptide in which one histidine in the amino acid sequence shown in SEQ ID NO:1 is replaced with aspartic acid or glutamic acid;
(1-2) A polypeptide in which one or two histidines in the amino acid sequence shown in SEQ ID NO:2 are replaced with aspartic acid or glutamic acid;
(1-3) A polypeptide in which 1 to 3 histidines in the amino acid sequence shown in SEQ ID NO: 3 are replaced with aspartic acid or glutamic acid;
(1-4) A polypeptide in which 1 to 4 histidines in the amino acid sequence shown in SEQ ID NO: 4 are replaced with aspartic acid or glutamic acid;
(1-5) A polypeptide in which 1 to 5 histidines in the amino acid sequence shown in SEQ ID NO:5 are replaced with aspartic acid or glutamic acid;
(1-6) A polypeptide in which 1 to 6 histidines in the amino acid sequence shown in SEQ ID NO:6 are replaced with aspartic acid or glutamic acid;
(1-7) A polypeptide in which 1 to 7 histidines in the amino acid sequence shown in SEQ ID NO: 7 are replaced with aspartic acid or glutamic acid;
(1-8) A polypeptide in which 1 to 8 histidines in the amino acid sequence shown in SEQ ID NO: 8 are replaced with aspartic acid or glutamic acid;
(1-9) A polypeptide in which 1 to 9 histidines in the amino acid sequence shown in SEQ ID NO: 9 are replaced with aspartic acid or glutamic acid;
(1-10) A polypeptide in which 1 to 10 histidines in the amino acid sequence shown in SEQ ID NO: 10 are substituted with aspartic acid or glutamic acid;
(1-11) A polypeptide in which 1 to 11 histidines in the amino acid sequence shown in SEQ ID NO: 11 are replaced with aspartic acid or glutamic acid;
The tag peptide is a tag peptide consisting of any one of the polypeptides (1-1) to (1-11) above, and hereinafter may be referred to as the "tag peptide of the present invention."

Another aspect of the present invention is
(2-1) mixing the present tag peptide fusion polypeptide with a carrier having one or more metal ions selected from nickel, copper, zinc, and cobalt immobilized thereon, thereby allowing the tag peptide fusion polypeptide to be adsorbed onto the carrier;
(2-2) washing the carrier onto which the tag peptide fusion polypeptide is adsorbed with a washing solution;
(2-3) eluting the tag peptide fusion polypeptide adsorbed to the carrier from the carrier using imidazole, L-histidine, Tris base, or water;
This is a method for purifying a polypeptide, comprising steps (2-1) to (2-3), and hereinafter may be referred to as the "method for purifying the polypeptide of the present invention."

In this specification, histidine may be represented as H, aspartic acid as D, glutamic acid as E, tryptophan as W, valine as V, threonine as T, arginine as R, methionine as M, leucine as L, proline as P, asparagine as N, lysine as K, glutamine as Q, isoleucine as I, phenylalanine as F, cysteine as C, tyrosine as Y, serine as S, glycine as G, and alanine as A.

In this specification, "polypeptide" refers to a peptide formed by linking five or more amino acid residues through peptide bonds. In addition, in this specification, "tag peptide" refers to an affinity tag that has affinity for one or more metal ions selected from nickel, copper, zinc, and cobalt.

The tag peptide of the present invention is not particularly limited as long as it is a tag peptide consisting of any of the above polypeptides (1-1) to (1-11), but any amino acid, preferably histidine, aspartic acid or glutamic acid, may be further linked to the N-terminus or C-terminus of the tag peptide by 1 to 5 residues, preferably 1 to 3 residues, more preferably 1 or 2 residues, and even more preferably 1 residue.

In the polypeptides (1-1) to (1-11) of the present specification, the position of the histidine to be substituted with aspartic acid or glutamic acid is not particularly limited, but it is preferable to substitute so that aspartic acid or glutamic acid is linked between the histidines. Specifically, it is preferable to include at least one of the tripeptides HEH shown in SEQ ID NO: 12, EHE shown in SEQ ID NO: 13, HDH shown in SEQ ID NO: 14, or DHD shown in SEQ ID NO: 15.

In addition, in the polypeptide of (1-2) or (1-3), HE, EH, HD, or DH is present at two or more positions, more preferably three or more positions; in the polypeptide of (1-4) or (1-5), HE, EH, HD, or DH is present at two or more positions, more preferably three or more positions, and even more preferably four or more positions; in the polypeptide of (1-6) or (1-7), HE, EH, HD, or DH is present at two or more positions, more preferably three or more positions, even more preferably four or more positions, and even more preferably five or more positions; In the case of the polypeptide of (1-8) or (1-9), HE, EH, HD, or DH may be present at two or more sites, more preferably three or more sites, even more preferably four or more sites, even more preferably five or more sites, and among these, six or more sites, and in the case of the polypeptide of (1-10) or (1-11), HE, EH, HD, or DH may be present at two or more sites, more preferably three or more sites, even more preferably four or more sites, even more preferably five or more sites, and among these, six or more sites, particularly preferably seven or more sites, and most preferably eight or more sites. Examples of the polypeptide of (1-7) include the amino acid sequence shown in SEQ ID NO: 16, which contains five HD sites, and the amino acid sequence shown in SEQ ID NO: 17, which contains five HE sites.

When multiple histidines are substituted, the combination of aspartic acid or glutamic acid that substitutes is not particularly limited, and may be all aspartic acid, all glutamic acid, or a combination of aspartic acid and glutamic acid.

The tag peptide fusion polypeptide of the present invention may be a tag peptide fusion polypeptide in which the above-mentioned tag peptide and a polypeptide are fused (hereinafter, also referred to as the "tag peptide fusion polypeptide of the present invention"). In such a tag peptide fusion polypeptide, any amino acid may be bound between the polypeptide to be fused and the tag peptide, or the polypeptide to be fused and the tag peptide may be directly linked. The any amino acid may be 1 to 3000 residues, preferably 2 to 1000 residues, and more preferably 3 to 100 residues.

Examples of the polypeptide to be fused with the tag peptide of the present invention include proteins, peptide hormones, epitopes, etc. Examples of the protein include, but are not limited to, enzymes, fluorescent proteins, structural proteins, metal binding proteins, cofactor proteins, antibodies, glycoproteins, etc.

Furthermore, the tag peptide fusion polypeptide may contain a tag sequence. The tag sequence may be located on either the N-terminus or C-terminus of the tag peptide, or on both sides. Examples of the tag sequence include a FLAG sequence, an HA sequence, a MYC sequence, a GFP sequence, an MBP sequence, a GST sequence, a SNAP sequence, an ACP sequence, a CLIP sequence, a TAP sequence, and a V5 sequence.

In addition, the tag peptide fusion polypeptide may contain a polypeptide consisting of an amino acid sequence that recognizes a protease between the tag peptide and the polypeptide. Examples of amino acid sequences that recognize a protease include polypeptides consisting of amino acid sequences that recognize thrombin, enterokinase, TEV protease, factor Xa, and PreScission (registered trademark). If a polypeptide consisting of an amino acid sequence that recognizes such a protease is provided, it becomes possible to cleave the fusion between the tag peptide and the polypeptide using a protease as necessary.

The above-mentioned tag peptide and the polypeptide fused to the tag peptide can be produced by known techniques such as genetic engineering techniques and peptide synthesis using liquid phase or solid phase methods.

The polynucleotide encoding the tag peptide of the present invention is not particularly limited as long as it is a polynucleotide that encodes the tag peptide of the present invention (hereinafter also referred to as the "polynucleotide of the present invention"), and such a polynucleotide can be prepared by a known method using genetic engineering techniques. Examples of the polynucleotide encoding the tag peptide of the present invention include the polynucleotides shown in SEQ ID NO: 18 or 19, but sequences in which the histidine, aspartic acid, or glutamic acid in these nucleotides are replaced with other corresponding codons may also be used.

The expression vector containing the polynucleotide of the present invention is not particularly limited as long as it is an expression vector containing the above-mentioned polynucleotide of the present invention (hereinafter also referred to as "the expression vector of the present invention"). The expression vector can be appropriately selected depending on the host, and examples thereof include bacterial expression vectors such as pET vectors, pBT vectors, pGEX vectors, pKK vectors, pSE vectors, and pQE vectors; mammalian expression vectors such as pcDNA vectors, pCMV-FLAG vectors, pEGFP vectors, and pcDM vectors; yeast expression vectors such as YEp vectors; insect expression vectors such as pFastBacI vectors and BakPAK vectors; and plant expression vectors such as tobacco mosaic virus vectors.

The method for purifying the polypeptide of the present invention includes the steps of:
(2-1) mixing a carrier having one or more metal ions selected from nickel, copper, zinc, and cobalt immobilized thereon with the present tag peptide fusion polypeptide in the presence of an adsorption solution to adsorb the tag peptide fusion polypeptide onto the carrier;
(2-2) washing the carrier onto which the tag peptide fusion polypeptide is adsorbed with a washing solution;
(2-3) eluting the tag peptide fusion polypeptide adsorbed to the carrier from the carrier using imidazole, L-histidine, Tris base, or water;
Any method for purifying a polypeptide comprising steps (2-1) to (2-3) above (hereinafter also referred to as the "method for purifying the polypeptide of the present invention") may be used, and the metal ion is preferably nickel.

By binding the metal ion to the tag peptide fusion polypeptide of the present invention, it becomes possible to immobilize the tag peptide fused polypeptide on the carrier. The tag peptide fused polypeptide and the carrier on which the metal ion is immobilized are coordinated immediately after mixing in the presence of an adsorption solution, but they may be allowed to stand for 1 minute or more, 1 hour or more, or 6 hours or more after mixing.

The above carrier is not particularly limited as long as it is insoluble in water, and examples of the carrier include silica gel, porous glass, cellulose, agarose, dextran, polyacrylamide, and magnetic beads.

The adsorption solution is not particularly limited as long as it is capable of mixing the carrier on which the metal ions are immobilized with the present tag peptide fusion polypeptide, binding the metal ions immobilized on the carrier with the present tag peptide fusion polypeptide, and allowing the present tag peptide fusion polypeptide to be adsorbed to the carrier, and examples of the adsorption solution include solutions containing alkali metal salts such as potassium, sodium, lithium, rubidium, etc., solutions containing alkaline earth metal salts such as calcium, magnesium, etc., and buffers such as phosphate buffer, Tris buffer, etc. Examples of the solutions containing alkali metal salts include KCl, NaCl, LiCl, and RbCl solutions, and examples of the solutions containing alkaline earth metal salts include _CaCl2 and _MgCl2 solutions.

When a solution containing an alkali metal salt or an alkaline earth metal salt is used as the adsorption solution, the concentration can be 0.03 to 2.5 M, 0.05 to 2.2 M, 0.1 to 2 M, 0.5 to 1.8 M, or 1 to 1.5 M. By making the concentration 0.03 M or higher, it is possible to increase the adsorption efficiency between the metal ions immobilized on the carrier and the tag peptide fusion polypeptide of the present invention.

When a buffer solution is used as the adsorption solution, the pH can be adjusted as appropriate depending on the type and concentration of the buffer solution used, but examples of the pH include pH 6 to 10, preferably pH 6.5 to 9, and more preferably pH 7 to 8.6. The concentration of the buffer solution can be 0.05 to 2 M, and preferably 0.1 to 1 M.

The washing solution is not particularly limited as long as it maintains or almost maintains the bond between the metal ion immobilized on the carrier and the tag peptide fusion polypeptide of the present invention. Here, "a solution in which the bond between the metal ion immobilized on the carrier and the tag peptide fusion polypeptide of the present invention is almost maintained" means a solution in which, even if the bond between the metal ion immobilized on the carrier and the tag peptide fusion polypeptide of the present invention is once released, the metal ion can re-bind to the metal ion immobilized on the surrounding carrier (lower in the case of using a column).

The above-mentioned adsorption solution can be used as the washing solution. When a solution containing an alkali metal salt or a solution containing an alkaline earth metal salt is used as the washing solution, the concentration can be 0.03 to 2.5 M, 0.05 to 2.2 M, 0.1 to 2 M, 0.5 to 1.8 M, or 1 to 1.5 M. However, from the viewpoint of preventing non-specific binding of non-target polypeptides to metal ions immobilized on the carrier, the lower limit of the concentration when a solution containing an alkali metal salt or a solution containing an alkaline earth metal salt is used can be 0.07 M, preferably 0.05 M, and more preferably 0.03 M, and the upper limit can be 0.1 M, preferably 0.08 M, and more preferably 0.06 M.

When a buffer solution is used as the washing solution, the pH can be adjusted as appropriate depending on the type and concentration of the buffer solution used, but examples of the pH include pH 6 to 10, preferably pH 6.5 to 9, and more preferably pH 7 to 8.6. Examples of the concentration of the buffer solution include 0.05 to 2 M, and preferably 0.1 to 1 M. However, from the viewpoint of preventing non-specific binding of unintended polypeptides to metal ions immobilized on the carrier, examples of the pH include pH 7 to 8, and preferably pH 7.2 to 7.4.

The elution solution is not particularly limited as long as it is a solution that can release the bond between the metal ion immobilized on the carrier and the tag peptide fusion polypeptide of the present invention, but examples of the elution solution include an imidazole solution, an L-histidine solution, a buffer solution, an alkali metal salt solution, and water.

When an imidazole solution is used as the eluent, the concentration is preferably 0.005 to 1 M, more preferably 0.05 to 0.1 M. Elution may be performed using an imidazole solution of a predetermined concentration, but elution may also be performed using a concentration gradient of, for example, 0-100 mM imidazole.

When an L-histidine solution is used as the eluent, the concentration is preferably 0.005 to 1 M, more preferably 0.05 to 0.1 M. Elution may be performed using an L-histidine solution of a predetermined concentration, but elution may also be performed using a concentration gradient of, for example, 0-100 mM histidine.

When a buffer solution is used as the eluent, the pH can be adjusted as appropriate depending on the type and concentration of the buffer solution used, but is preferably pH 6 to 10, and more preferably pH 8.5 to 9. The concentration of the buffer solution can be 0.1 to 2 M, and preferably 0.5 to 1 M.

The kit for purifying the polypeptide of the present invention may contain the expression vector of the present invention, and may also contain a carrier on which one or more metal ions selected from nickel, copper, zinc, and cobalt are immobilized. If necessary, the kit may also contain instructions for purifying the polypeptide, and an adsorption solution, a washing solution, and an elution solution used in the method for purifying the polypeptide of the present invention.

The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[Example 1]
Although histidine tags consisting of six histidines have been widely used, it is known that the use of such a tag with consecutive histidines reduces the activity of the protein. Therefore, we investigated whether the reduction in protein activity could be suppressed by combining histidine with an amino acid other than histidine. We produced a red fluorescent protein (RFP protein) with a tag peptide with consecutive histidines fused to the C-terminus of the RFP protein, and a tag peptide with various amino acids arranged between three histidines. We produced a strain that produces RFP fused with such a tag peptide, and evaluated the activity of the protein by examining the fluorescence of RFP.

(a-1) 6H: HHHHHH (SEQ ID NO: 2)
(a-2) 12H: HHHHHHHHHHHH (SEQ ID NO: 8)
(a-3) 15H: HHHHHHHHHHHHHHHH (SEQ ID NO: 11)
(a-4) 18H: HHHHHHHHHHHHHHHHHHH (SEQ ID NO: 20)
(a-5) 3HW × 5: HHHWHHHHWHHHHWHHHH (SEQ ID NO: 21)
(a-6) 3HV × 5: HHHVHHHVHHHVHHHVHHH (SEQ ID NO: 22)
(a-7) 3HT × 5: HHHTHHHHHHHHHHHHHH (SEQ ID NO: 23)
(a-8) 3HR × 5: HHHRHHRHHRHHRHHRHHHH (SEQ ID NO: 24)
(a-9) 3HM × 5: HHHMHHHMHHHMHHHMHHHH (SEQ ID NO: 25)
(a-10) 3HL x 5: HHHLHHHLHHHLHHHLHHH (SEQ ID NO: 26)
(a-11) 3HP × 5: HHHPHHHPHHHHHHHHHH (SEQ ID NO: 27)
(a-12) 3HN × 5: HHHNHHHNHHHNHHHNHHH (SEQ ID NO: 28)
(a-13) 3HK × 5: HHHKHHHKHHHKHHHKHHH (SEQ ID NO: 29)
(a-14) 3HQ × 5: HHHQHHHQHHHQHHHQHHH (SEQ ID NO: 30)
(a-15) 3HI × 5: HHHIHHHIHHHIHHHIHHH (SEQ ID NO: 31)
(a-16) 3HF × 5: HHHFHHHFHHHFHHHFHHH (SEQ ID NO: 32)
(a-17) 3HE × 5: HHHEHHHEHHHEHHHEHHHH (SEQ ID NO: 33)
(a-18) 3HD × 5: HHHDHHDHHDHHDHHDHHHH (SEQ ID NO: 34)
(a-19) 3HC × 5: HHHCHHHCHHHWHHHCHHH (SEQ ID NO: 35)
(a-20) 3HY × 5: HHHYHHHYHHHYHHHYHHH (SEQ ID NO: 36)
(a-21) 3HS × 5: HHHSHHSHHSHHSHHSHH (SEQ ID NO: 37)
(a-22) 3HG × 5: HHGHHHHGHHHHHGHHHH (SEQ ID NO: 38)
(a-23) 3HA × 5: HHHAHHHAHHHAHHHAHHH (SEQ ID NO: 39)

Using the YEpGAPcherry vector (see JP 2017-88536 A) as a template, primers were designed to add the tag peptide to the C-terminus of the RFP protein, and PCR was performed to amplify the base sequence encoding the tag peptide-fused RFP. The PCR product was inserted into the YEpGAPcherry vector by homologous recombination and transformed into the BY4741 strain of Saccharomyces cerevisiae (see Brachmann et al., Yeast Volume 14, Issue 2, 30 January 1998), to produce transformants that produce RFPs fused with the respective tag peptides.

Next, 1 mL of YPD was added to a 24-well plate, and the transformant prepared above was inoculated and cultured at 28°C and 150 rpm for 24 hours. After that, 10 μL of the culture solution was taken and mixed with 1 mL of YPD, and cultured at 28°C and 150 rpm for another 24 hours. 10 μL of the culture solution was diluted 100-fold with distilled water, and OD600 and fluorescence (excitation 558, emission 613) were measured using a Synergy MX SMATBL (BioTek). RFP activity (intensity of red color) was calculated as "RFP activity = fluorescence / OD600". The results are shown in Figure 1.

As shown in Figure 1, the activity of RFP decreased as the number of consecutive histidines increased. In addition, it was revealed that the activity of RFP was 1.5 times higher when there were proline (P), glutamic acid (E), aspartic acid (D), glycine (G), alanine (A), asparagine (N), and glutamine (Q) between three consecutive histidines, compared to a tag with the same 15 histidines.

[Example 2]
It was confirmed from Example 1 that the decrease in protein activity was suppressed by including proline (P), glutamic acid (E), aspartic acid (D), glycine (G), alanine (A), asparagine (N), or glutamine (Q) in consecutive histidines. Therefore, the activity of the protein was examined in various combinations of histidine and aspartic acid shown below.

(b-1) No tag peptide (b-2) HHHHHH (SEQ ID NO: 2)
(b-3) HDHDHDHDHDH (SEQ ID NO: 16)
(b-4) HHDHHDHH (SEQ ID NO: 40)
(b-5) HHHDHHH (SEQ ID NO: 41)
(b-6) HHHHHHDDDDDDD (SEQ ID NO: 42)
(b-7) DDDDDDHHHHHH (SEQ ID NO: 43)
(b-8) DDDHHHHHHDDD (SEQ ID NO: 44)
(b-9) HHHHHHDDD (SEQ ID NO: 45)
(b-10) DDDHHHHHH (SEQ ID NO: 46)

A transformant was prepared that produced tag peptide-fused GLuc in which the tag peptides (b-1) to (b-10) were added to the C-terminus of Gaussia luciferase (GLuc), and the activity of the protein was evaluated by examining the fluorescence of GLuc.

Using a vector containing GLuc (New England Biolabs Japan) as a template, primers were designed to add the tag peptide to the C-terminus of the GLuc protein, and PCR was performed to amplify the base sequence encoding the tag peptide-fused GLuc. The PCR product was incorporated into the YEpGAPcherry vector by homologous recombination and transformed into the BY4741 strain of Saccharomyces cerevisiae, producing transformants that produce GLuc fused with each tag peptide.

Next, 1 mL of YPD was added to a 24-well plate, and the transformant prepared above was inoculated and cultured at 28°C and 150 rpm for 24 hours. Then, 10 μL of the culture solution was taken, mixed with 1 mL of YPD, and cultured at 28°C and 150 rpm for another 24 hours. 10 μL of the culture solution was diluted 100-fold with distilled water, and the OD600 was measured using a spectrophotometer. In addition, the relative expression level of luciferase in the culture solution (RLU/(μL·OD)) was calculated as luciferase activity using BioLux (registered trademark) Gaussia Luciferase Assay Kit (New England Biolabs) and a luminometer (Glo Max20/20n Luminometer: Promega). The results are shown in Figure 2.

As is clear from FIG. 2, luciferase activity is more than doubled when aspartic acid (D) is contained compared to 6H, which is a sequence of six consecutive histidines (H). In particular, HDHDHDHDHDHDH, which is an alternating sequence of histidines and aspartic acids as shown in SEQ ID NO: 16, was confirmed to have activity more than 10 times higher than 6H. Therefore, it was believed that high luciferase activity could be maintained by reducing the number of consecutive histidines and/or the number of consecutive aspartic acids.

[Example 3] <Adsorption to nickel beads in a tube-1>
It was confirmed in Example 2 that the use of HDHDHDHDHDDHDH (6HD) consisting of the amino acid sequence shown in SEQ ID NO: 16 as a tag peptide suppresses the decrease in protein activity. In order to purify a protein using a tag peptide, the tag peptide must be adsorbed to nickel. Thus, the adsorption of the tag peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 (HDHDHDHDHDDHDH: 6HD) or the tag peptide consisting of the amino acid sequence shown in SEQ ID NO: 17 (HEHEHEHEHEH: 6HE) to nickel beads was examined.

(Preparation of RFP protein extract)
As in Example 1 above, a tag peptide fusion polypeptide (RFP-6HD) in which a tag peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 is fused to the C-terminus of the RFP protein, or a tag peptide fusion polypeptide (RFP-6HE) in which a tag peptide consisting of the amino acid sequence shown in SEQ ID NO: 17 is fused to the C-terminus of the RFP protein, was produced using the YEpGAPcherry vector. The transformant, the transformant producing RFP-6H produced in Example 1, and the transformant producing RFP without a tag peptide were cultured, the culture solution was transferred to a 15 mL tube, centrifuged at 3000 rpm for about 1 minute, and the supernatant was discarded. 5 mL of 0.1 M Tris base was added thereto, and after stirring, the mixture was centrifuged at 3000 rpm for 1 minute and the supernatant was discarded. 1.5 mL of 0.1 M Tris base + 1% Triton X-100 was added thereto, and after stirring, the mixture was heated at 50° C. for 1 hour. Then, the mixture was centrifuged at 9000 rpm for 5 minutes, and the supernatant was used as a protein extract of RFP-6HD, RFP-6HE, RFP-6H, or RFP.

(Adsorption onto nickel beads)
Nickel beads (His-Select Nickel Affinity Gel: Sigma-Aldrich) 50 μL, the above RFP-6HE, RFP-6HD, RFP-6H or RFP protein extract 100 μL, and adsorption solution (0.1 M Tris-HCl pH 7) 100 μL were added to a 1.5 mL microcentrifuge tube (Sorenson) and mixed, and mixed at 30 rpm for 10 minutes using an Intelli-mixer, and then centrifuged at 6000 rpm for several seconds. The results are shown in FIG. 3.

As is clear from FIG. 3, it was confirmed that RFP-6HE and RFP-6HD, which are fused with 6HE and 6HD as tag peptides, were adsorbed to nickel beads.
[Example 4] <Adsorption to nickel beads in a tube-2>

(Effect of salt)
Although Tris-HCl was used as the adsorption solution in Example 3, other adsorption solutions were used to examine the adsorption of the tag peptide fusion peptide to nickel beads. In addition, although a protein extract produced in yeast was used as the tag peptide fusion peptide in Example 3, RFP-6HE and RFP-6H protein extracts produced in E. coli by the following method were used.

The transformant RAK26118 strain (DH5α strain/genotype: pAmpOri-srlA100p-eEmRFP-6HE: Nakamura et al., molecular Biotechnology December 2018, Volume 60, Issue 12, pp 912-923) expressing RFP-6HE with 6HE bound to the C-terminus of RFP, and the transformant RAK26117 strain (DH5α strain/genotype: pAmpOri-srlA100p-eEmRFP-6H) expressing RFP-6H with 6H bound to the C-terminus of RFP were cultured at 37°C, 48 hours, and 175 rpm in a medium containing yeast extract and polypeptone. Next, the culture solution was transferred to a 50 mL tube, centrifuged at 12,000 rpm for 1 minute, and the supernatant was discarded. 3 mL of a solution containing 1% Triton X-100 was added and stirred to obtain RFP-6HE or RFP-6H protein extract.

The adsorption solution was also tested using 0.1M LiCl, 0.1M NaCl, and 0.1M KCl, and with concentrations of 1M KCl, 0.1M KCl, and 0.01M KCl. The results for each adsorption solution are shown in Figure 4(a) and (b), respectively.

As is clear from Figures 4(a) and (b), it was revealed that RFP-6HE was adsorbed to nickel beads in the presence of any of the salts 0.1M LiCl, NaCl, and KCl. When distilled water was used as the adsorption solution, RFP-6HE was not adsorbed to nickel beads (not shown). In addition, in KCl, RFP-6HE was not adsorbed at a salt concentration of 0.01M, but was adsorbed at 0.1M and 1M. From these results, it was confirmed that RFP-6HE is adsorbed to nickel beads in the presence of salt at a certain concentration or higher. On the other hand, RFP-6H was adsorbed even with distilled water and 0.01M KCl, and it was considered that RFP-6H is adsorbed to nickel beads regardless of the salt concentration. In addition, since a KCl solution with a concentration of 1M is usually the salt concentration used for elution in normal ion exchange chromatography, if RFP-6HE is adsorbed to nickel using a KCl solution with a concentration of 1M, it is possible to reduce nonspecific adsorption of untargeted proteins.

In addition, since adsorption occurred in the presence of a certain amount of salt, it was suggested that the side chain of glutamic acid may be involved in the adsorption of RFP-6HE to nickel beads.

[Example 5] <Elution of RFP-6HE (tube)>
It was confirmed from the above Examples 3 and 4 that RFP-6HE is adsorbed to nickel beads. In order to purify the protein, it is necessary to elute the protein adsorbed to the nickel beads. Therefore, it was further examined using a microcentrifuge tube whether the adsorbed protein can be eluted from the nickel beads.

(Elution with imidazole)
6HE was adsorbed onto nickel beads in the same manner as in Example 3, using 2 M KPO ₄ pH 8.0 as the adsorption solution. Next, elution was performed with 0.1 M imidazole. The results are shown in Figure 5. As is clear from Figure 5, the entire tube turned blue-purple, and it was confirmed that RFP-6HE was eluted from the nickel beads with 0.1 M imidazole, similar to RFP-6H.

(Elution with histidine)
Elution by imidazole was confirmed, but since imidazole is basic, it may be necessary to remove imidazole from the elution solution depending on the type of protein to be eluted and the use of the eluted protein. Here, imidazole is structurally partially similar to histidine. Histidine is also less expensive than imidazole and the pH can be easily adjusted. Therefore, elution was attempted with histidine (1, 10, 20, 40, 60, 80, 100 mM) instead of imidazole. The results are shown in FIG. 6. As is clear from FIG. 6, it was confirmed that RFP-6HE was also eluted from nickel beads by histidine.

(Elution with Tris buffer)
In the elution of histidine tag using imidazole, Tris buffer is often used to control pH. Therefore, RFP-6HE was adsorbed to nickel beads in the same manner as in Example 3 using 2M KPO ₄ pH 8.0 as the adsorption solution. Next, elution was performed with Tris buffer (pH 8, 9, and 10, concentration 0.5 and 1M). The results are shown in FIG. 7. As is clear from FIG. 7, it was found that RFP-6HE was eluted from nickel beads with any Tris buffer, and that elution was easier with 1M than with 0.5M. It was revealed that RFP-6H was difficult to elute at 0.5M, whereas RFP-6HE could be eluted, and was particularly easy to elute at pH 9 and 10.

[Example 6] <Elution of RFP-6HE (column)>
In Example 5, elution was performed using a tube with a predetermined elution solution, but elution using a column is common in protein purification, so elution using a column was examined.

A nickel-filled column (His Trap HP: GE Healthcare Japan) was connected to a peristaltic pump UV monitor integrated chromatography system AKTA start (GE Healthcare Japan). RFP-6HE or RFP-6H was applied to the nickel-filled column, adsorbed with 1M KPO ₄ pH 8.0, washed with the same solution for 10 minutes, and further washed with distilled water for 5 minutes. Next, RFP-6HE or RFP-6H was eluted with a concentration gradient of 0-100 mM imidazole to measure UV280 nm absorption. The liquid in which a peak was observed upon elution was analyzed by SDS-PAGE. The gel was electrophoresed at 300 V for 35 minutes using Extra PAGE One Precast Gel 10-20%. The results of elution using a concentration gradient of imidazole are shown in Figures 8(a) and (b), and the results of SDS-PAGE analysis are shown in Figure 8(c).

As is clear from Figures 8(a) and (b), RFP-6HE elutes with a sharper peak than RFP-6H, and can be eluted with a low concentration of imidazole. In addition, as is clear from Figure 8(c), it was confirmed that RFP-6HE was purified to the same extent as RFP-6H.

In Example 1, since the activity of the protein was high when the tag peptide contained proline (P) and glycine (G) between three consecutive histidines, RFP-6HP and RFP-6HG were prepared in the same manner as in Example 2, and elution from a nickel-packed column was attempted using a concentration gradient of 0-100 mM imidazole as described above. The results are shown in Figures 9(a) and (b).

As is clear from Figures 9(a) and (b), RFP-6HP and RFP-6HG were hardly eluted from the nickel-packed column, demonstrating that RFP-6HE is preferable for purifying proteins, rather than RFP-6HP and RFP-6HG.

[Example 7] <Elution of RFP-6HE (column)-2>
In Example 6, washing with distilled water was performed before elution with the imidazole concentration gradient, but subsequent analysis by the present inventors confirmed that some of the product was also eluted with distilled water. Therefore, in order to further improve the degree of purification, an elution method was investigated in which the washing step with distilled water was omitted from the elution method in Example 6. The results are shown in FIG.

As is clear from Figure 10, the elution of RFP-6HE became sharper compared to Figure 8. Therefore, it became clear that it is better not to use water for washing before elution.

[Example 8] <Washing of RFP-6HE (column)>
The results of Example 4 show that RFP-6HE is less likely to adsorb to nickel depending on the salt concentration. On the other hand, in order to increase the degree of purification, a low salt concentration is preferable as a washing solution in order to prevent non-specific binding of non-target proteins to nickel. Therefore, washing was performed using a concentration gradient of NaCl, KCl, LiCl, and RbCl, and the concentration of the salt solution was examined when washing was performed.

RFP-6HE was applied to a nickel-filled column in the same manner as in Example 6, and after adsorption with 1M KPO ₄ pH 8.0, it was washed with a concentration gradient of 1-0.1M NaCl, 1-0.1M KCl, or 1-0.1M LiCl, and a concentration gradient of 0.1-0.01M NaCl, 0.1-0.01M KCl, 0.1-0.01M LiCl, or 0.1-0.01M RbCl, and UV280nm was measured. The results for NaCl are shown in Figure 11, and the results for KCl, LiCl, and RbCl are shown in Figures 12(a) to (c).

As shown in Figures 11 and 12, none of NaCl, KCl, LiCl, or RbCl was eluted at a concentration of 0.1 M. On the other hand, movement within the nickel column was observed at 0.01 M. Therefore, from the viewpoint of improving the degree of purification and the yield in protein purification, it was considered appropriate to use a salt solution with a concentration of 0.03 M or more for washing.

[Example 9] <Elution of RFP-6HE (column)-3>
It was confirmed from Example 8 that when washing was performed using NaCl, KCl, LiCl, and RbCl, RFP-6HE remained adsorbed to the nickel column at approximately 0.03 M. Therefore, after the above washing, it was examined whether elution was possible with imidazole, histidine, or water.

RFP-6HE was applied to a nickel-filled column in the same manner as in Example 6, and after adsorption with 1 M KPO ₄ pH 8.0, it was washed with 0.05 M KCl for 30 minutes and then eluted with a concentration gradient of 0-100 mM imidazole or L-histidine. The results are shown in Figures 13 and 14.

As is clear from Figure 13, a sharp peak was observed at low concentrations (5-20 mM) of imidazole. Also, as is clear from Figure 14, a sharp elution was observed at low concentrations (5-15 mM) of L-histidine.

Furthermore, RFP-6HE was applied to a nickel-packed column in the same manner as in Example 6, and after adsorption with 1 M KPO ₄ pH 8.0, it was washed with 0.1 M KCl for 50 minutes and then eluted with distilled water for about 30 minutes. The results are shown in FIG.

As is clear from Figure 15, after washing with low-concentration salt, sharp elution occurs even with water.

[Example 10] <Elution of DNA polymerase (column)>
In the above, RFP was purified, but purification using another protein, DNA polymerase, was also examined.

A transformant of E. coli HST08 strain that produces a tag peptide fusion DNA polymerase in which the tag peptide 6HE is added to the C-terminus of the DNA polymerase was prepared in the same manner as in Example 2. Next, a protein extract of DNA polymerase-6HE was prepared in the same manner as in Example 3, except that 3 mL of 0.1 M Tris-HCl pH 8.0 and 1% Triton X-100 was added and the hot bath after stirring was set to 95°C for 5 minutes.

The DNA polymerase-6HE was applied to a nickel-packed column in the same manner as in Example 6, and after adsorption with 1M KPO ₄ pH 8.0, it was washed with 1M KCl for 30 minutes and eluted with 100 mM imidazole for about 30 minutes, and UV at 280 nm was measured. The results are shown in FIG.

As is clear from Figure 16, it was confirmed that DNA polymerase-6HE eluted extremely sharply. Therefore, it became clear that by using this tag peptide, it is possible to purify proteins to a high degree of purity regardless of the type of protein.

Claims (14)

A tag peptide consisting of any one of the following polypeptides ( 1-3' ) to (1-9 ' ) :
( 1-3 ' ) In the amino acid sequence shown in SEQ ID NO: 3, two or three histidines are replaced with aspartic acid or glutamic acid , and
a polypeptide containing two or three histidine-aspartic acid or histidine-glutamic acid residues, or two or three aspartic acid-histidine or glutamic acid-histidine residues ;
(1-4 ' ) Two or three histidines in the amino acid sequence shown in SEQ ID NO: 4 are replaced with aspartic acid or glutamic acid , and
a polypeptide containing two or three histidine-aspartic acid or histidine-glutamic acid residues, or two or three aspartic acid-histidine or glutamic acid-histidine residues ;
(1-5 ' ) Three histidines in the amino acid sequence shown in SEQ ID NO:5 are replaced with aspartic acid or glutamic acid , and
a polypeptide containing three histidine-aspartic acid or three histidine-glutamic acid residues, or three aspartic acid-histidine or glutamic acid-histidine residues ;
(1-6 ' ) Three or four histidines in the amino acid sequence shown in SEQ ID NO:6 are replaced with aspartic acid or glutamic acid , and
a polypeptide containing three or four histidine-aspartic acid or histidine-glutamic acid units, or three or four aspartic acid-histidine or glutamic acid-histidine units ;
(1-7 ' ) 4 or 5 histidines in the amino acid sequence shown in SEQ ID NO: 7 are replaced with aspartic acid or glutamic acid , and
a polypeptide containing four or five histidine-aspartic acid or histidine-glutamic acid residues, or four or five aspartic acid-histidine or glutamic acid-histidine residues ;
(1-8 ' ) 4 or 5 histidines in the amino acid sequence shown in SEQ ID NO: 8 are replaced with aspartic acid or glutamic acid , and
a polypeptide containing four or five histidine-aspartic acid or histidine-glutamic acid residues, or four or five aspartic acid-histidine or glutamic acid-histidine residues ;
(1-9 ' ) 5 or 6 histidines in the amino acid sequence shown in SEQ ID NO:9 are replaced with aspartic acid or glutamic acid , and
a polypeptide containing five or six histidine-aspartic acid or histidine-glutamic acid units, or five or six aspartic acid-histidine or glutamic acid-histidine units ; The tag peptide according to claim 1 , which comprises a tripeptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 12 to 15. The tag peptide according to claim 1 or 2, which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17. A tag peptide fusion polypeptide in which the tag peptide according to any one of claims 1 to 3 is fused with a polypeptide. The tag peptide fusion polypeptide according to claim 4 , wherein the polypeptide fused to the tag peptide is a protein. A polynucleotide encoding the tag peptide according to any one of claims 1 to 5 . An expression vector containing the polynucleotide of claim 6 . A method for purifying a polypeptide, comprising the following steps (2-1) to (2-3):
(2-1) a step of mixing a carrier having one or more metal ions selected from nickel, copper, zinc, and cobalt immobilized thereon with the tag peptide fusion polypeptide according to claim 4 or 5 in the presence of an adsorption solution to adsorb the tag peptide fusion polypeptide onto the carrier;
(2-2) washing the carrier onto which the tag peptide fusion polypeptide is adsorbed with a washing solution;
(2-3) eluting the tag peptide fusion polypeptide adsorbed to the carrier from the carrier using an elution solution; 9. The method for purifying a polypeptide according to claim 8 , wherein in the step (2-1), the adsorption solution is an alkali metal salt solution of 0.03 M or more. 10. The method for purifying a polypeptide according to claim 8 or 9 , wherein in the step (2-2), the washing solution is an alkali metal salt solution of 0.03 M or more. The method for purifying a polypeptide according to any one of claims 8 to 10 , wherein in the step (2-3), the eluent is an imidazole solution, an L-histidine solution, a buffer solution, an alkali metal salt solution or water. The method for purifying a polypeptide according to any one of claims 8 to 11 , wherein the metal ion is nickel. A kit for purifying a polypeptide, comprising the expression vector according to claim 7 . (2-1') mixing a carrier having one or more metal ions selected from nickel, copper, zinc, and cobalt immobilized thereon with a tag peptide fusion polypeptide in the presence of an adsorption solution to adsorb the tag peptide fusion polypeptide onto the carrier;
(2-2') washing the carrier onto which the tag peptide fusion polypeptide is adsorbed with a washing solution;
(2-3') eluting the tag peptide fusion polypeptide adsorbed on the carrier from the carrier using an L-histidine solution, a buffer solution (excluding buffers containing imidazole) , or water;
The method for purifying a polypeptide comprising the steps (2-1') to (2-3'), wherein the tag fusion peptide in the step (2-1') is
( 1-4″ ) Four histidines in the amino acid sequence shown in SEQ ID NO:4 are replaced with aspartic acid or glutamic acid , and
A tag peptide consisting of a polypeptide containing four histidine-aspartic acid or histidine-glutamic acid residues, or four aspartic acid-histidine or glutamic acid-histidine residues ; or ( 1-5'' ) an amino acid sequence shown in SEQ ID NO:5 in which four histidine residues are replaced with aspartic acid or glutamic acid , and
The above purification method, wherein the polypeptide is a tag peptide fusion polypeptide in which the polypeptide is fused with a tag peptide consisting of a polypeptide containing four sites of histidine-aspartic acid or histidine-glutamic acid, or four sites of aspartic acid-histidine or glutamic acid-histidine.