Tag peptide and method for purifying polypeptide

Abstract

To provide a tag peptide that is capable of bonding one or more metal ions selected from nickel, copper, zinc and cobalt, and is capable of eluting even if liquid other than imidazole is used.SOLUTION: Polypeptide or the like that consists of an amino acid sequence shown by sequence number 16 or sequence number 17 is fabricated. Further, a method for purifying polypeptide comprising: (2-1) a process of mixing a carrier in which one or more metal ions selected from nickel, copper, zinc and cobalt are immobilized and a tag peptide-fused polypeptide including the tag peptide that consists of an amino acid sequence shown by sequence number 16 or sequence number 17 under the presence of adsorption liquid, and allowing the tag peptide-fused polypeptide to be adsorbed on the carrier; (2-2) a process of cleaning the carrier onto which the tag peptide-fused polypeptide was adsorbed by cleaning fluid; and (2-3) a process of eluting the tag peptide-fused polypeptide adsorbed onto the carrier from the carrier by using eluent.SELECTED DRAWING: None

Description

[Technical field]

[0001] 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. [Background technology]

[0002] When protein synthesis is performed using E. coli or yeast based on knowledge of genetic engineering, the polypeptide such as the target protein is often separated and purified. A widely used protein purification method is to use 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.

[0003] (1) A target protein tagged with six consecutive histidines is produced in host cells 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 5-10 mM) imidazole solution is passed through the column to wash it. 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.

[0004] However, since many proteins containing a lot of histidine and proteins that bind to nickel metal are present in the cells of E. coli and yeast in which protein synthesis has been performed, these proteins are also adsorbed to the nickel column, making it difficult to purify the target protein with high purity. In addition, when removing the target protein adsorbed to nickel from the nickel with an eluent, an imidazole aqueous solution is usually used, but when using the target protein, it was sometimes necessary to remove the imidazole.

[0005] As tag peptides, in addition to six consecutive histidines, 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 to 10; y is 0 to 4; z is 0 to 10] (see Patent Document 1), a peptide represented by 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 sequence of 30 amino acids) (see Patent Document 2), and a sequence of three or more consecutive basic amino acids (see Patent Document 3) have been disclosed. However, elution is mainly performed with imidazole, and there was no tag peptide that could be eluted with a solvent other than imidazole with high accuracy as necessary.

[0006] The present 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, so it is possible to immobilize a target protein with high purity. However, it has been difficult to elute the target protein as needed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 6-157600 [Patent Document 2] Japanese Patent Publication No. 63-251095 [Patent Document 3] JP 2008-241560 A [Patent Document 4] JP 2017-88536 A Summary of the Invention [Problem to be solved by the invention]

[0008] An object 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. [Means for solving the problem]

[0009] 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 with peptide tags. In the process, we have confirmed that the addition of consecutive histidines to a protein reduces the activity of the protein. Therefore, we investigated histidine tag peptides that can suppress the decrease in protein activity.

[0010] 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 and a highly active protein can be obtained, compared to the case where a tag peptide of consecutive histidines is used. 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.

[0011] 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted with aspartic acid or glutamic acid; [2] The tag peptide according to [1] above, characterized in that it contains 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) mixing a carrier having one or more metal ions selected from nickel, copper, zinc, and cobalt immobilized thereon with the tag peptide fusion polypeptide described in [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 liquid is an alkali metal salt solution 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 elution solution 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 described in [7] above. Effect of the Invention

[0012] By using the tag peptide of the present invention, after a 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. [Brief description of the drawings]

[0013] [Figure 1] FIG. 2 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. [Diagram 2] 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). [Diagram 3] 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. [Figure 4] FIG. 13 shows the results of investigating adsorption in Example 4 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. [Diagram 5] FIG. 13 shows the results of eluting RFP-6HE and RFP-6H adsorbed to nickel beads with imidazole in Example 5. [Figure 6] FIG. 13 shows the results of eluting RFP-6HE and RFP-6H adsorbed to nickel beads with histidine in Example 5. [Figure 7] FIG. 13 shows the results of eluting RFP-6HE and RFP-6H adsorbed to nickel beads with a Tris buffer solution in Example 5. [Figure 8]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 UV280 nm measurement, and Figure 8(c) shows the result of SDS-PAGE analysis. [Figure 9] 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. [Figure 10] 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. [Figure 11] 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 UV280 nm when washed with a concentration gradient of 0.1 M-0 M NaCl. [Figure 12] 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 of 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. [Figure 13] In Example 9, RFP-6HE adsorbed on a nickel column in 1 M KPO4, 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. [Figure 14] In Example 9, RFP-6HE adsorbed on a nickel column in 1 M KPO4, 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. [Figure 15]In Example 9, RFP-6HE adsorbed on a nickel column in 1 M KPO4 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. [Figure 16] In Example 10, DNA polymerase-6HE adsorbed on a nickel column in 1 M KPO4 pH 8.0 was washed with 0.05 M KCl for 30 minutes, and then eluted with 100 mM imidazole for about 30 minutes, and UV at 280 nm was measured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted 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 substituted 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 present tag peptide."

[0015] Another aspect of the present invention is (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 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; This is a method for purifying a polypeptide, comprising steps (2-1) to (2-3) above, and hereinafter may be referred to as the "method for purifying the present polypeptide."

[0016] 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.

[0017] As used herein, the term "polypeptide" refers to a peptide formed by linking five or more amino acid residues through peptide bonds. In addition, as used herein, the term "tag peptide" refers to an affinity tag having affinity for one or more metal ions selected from nickel, copper, zinc, and cobalt.

[0018] The tag peptide of the present invention is not particularly limited as long as it is a tag peptide consisting of any of the polypeptides (1-1) to (1-11) above. The N-terminus or C-terminus of such a tag peptide may further comprise 1 to 5 residues, preferably 1 to 3 residues, more preferably 1 or 2 residues, and even more preferably 1 residue of any amino acid, preferably histidine, aspartic acid or glutamic acid, linked thereto.

[0019] In the polypeptides (1-1) to (1-11) herein, the position of the histidine to be substituted with aspartic acid or glutamic acid is not particularly limited, but it is preferable to substitute such that aspartic acid or glutamic acid is linked between histidines. Specifically, it is preferable to contain at least one tripeptide of 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.

[0020] In addition, in the polypeptide of (1-2) or (1-3), HE, EH, HD, or DH In the polypeptide of (1-4) or (1-5), HE, EH, HD, or DH in the polypeptide of (1-6) or (1-7), HE, EH, HD, or DH in the polypeptide of (1-8) or (1-9), HE, EH, HD, or DH In the polypeptide of (1-10) or (1-11), HE, EH, HD, or DH of It may contain 2 or more sites, more preferably 3 or more sites, even more preferably 4 or more sites, even more preferably 5 or more sites, among which 6 or more sites, particularly preferably 7 or more sites, and most preferably 8 or more sites. Examples of the (1-7) polypeptide include the amino acid sequence shown in SEQ ID NO: 16 which contains 5 HD sites, and the amino acid sequence shown in SEQ ID NO: 17 which contains 5 HE sites.

[0021] When multiple histidines are substituted, the combination of substituting aspartic acid or glutamic acid is not particularly limited, and may be all aspartic acid, all glutamic acid, or a combination of aspartic acid and glutamic acid.

[0022] The tag peptide fusion polypeptide of the present invention may be a tag peptide fusion polypeptide in which the above-mentioned tag peptide of the present invention is fused with a polypeptide (hereinafter, also referred to as "tag peptide fusion polypeptide of the present invention"), and 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.

[0023] 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.

[0024] Furthermore, the tag peptide fusion polypeptide may contain a tag sequence, which may be located on either the N-terminus or C-terminus of the tag peptide, or on both sides, and examples of the tag sequence include a FLAG sequence, an HA sequence, a MYC sequence, a GFP sequence, a MBP sequence, a GST sequence, a SNAP sequence, an ACP sequence, a CLIP sequence, a TAP sequence, and a V5 sequence.

[0025] Furthermore, 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 the amino acid sequence that recognizes a protease include a polypeptide consisting of an amino acid sequence that recognizes 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.

[0026] The present tag peptide and the polypeptide fused to the present tag peptide can be prepared by known techniques such as genetic engineering techniques or liquid-phase or solid-phase peptide synthesis.

[0027] The polynucleotide encoding the tag peptide of the present invention is not particularly limited as long as it is a polynucleotide encoding 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.

[0028] 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 of the expression vector include bacterial expression vectors such as pET vectors, pBT vectors, pGEX vectors, pKK vectors, pSE vectors, pQE vectors, etc.; mammalian expression vectors such as pcDNA vectors, pCMV-FLAG vectors, pEGFP vectors, pcDM vectors, etc.; yeast expression vectors such as YEp vectors; insect expression vectors such as pFastBacl vectors, BakPAK vectors, etc.; and plant expression vectors such as tobacco mosaic virus vectors.

[0029] 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 the 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.

[0030] The binding of the metal ion to the tag peptide fusion polypeptide of the present invention enables the tag peptide-fused polypeptide to be immobilized on the carrier. The tag peptide-fused polypeptide of the present invention and the carrier on which the metal ion is immobilized are coordinate-bonded immediately after mixing in the presence of an adsorption solution, but may be left to stand for 1 minute or more, 1 hour or more, or 6 hours or more after mixing.

[0031] The 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.

[0032] 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.

[0033] 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 setting the concentration to 0.03 M or more, it is possible to increase the adsorption efficiency between the metal ion immobilized on the carrier and the present tag peptide fusion polypeptide.

[0034] When a buffer solution is used as the adsorption solution, the pH can be appropriately adjusted depending on the type and concentration of the buffer solution used, and may be from pH 6 to 10, preferably from pH 6.5 to 9, and more preferably from pH 7 to 8.6. The concentration of the buffer solution may be from 0.05 to 2 M, and preferably from 0.1 to 1 M.

[0035] The washing solution is not particularly limited as long as it is 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 maintained or almost maintained. 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)

[0036] 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.

[0037] When a buffer solution is used as the washing solution, the pH can be appropriately adjusted depending on the type and concentration of the buffer solution used, and may be pH 6 to 10, preferably pH 6.5 to 9, and more preferably pH 7 to 8.6. The concentration of the buffer solution may be 0.05 to 2 M, and preferably 0.1 to 1 M. However, from the viewpoint of preventing non-target polypeptides from non-specifically binding to metal ions immobilized on the carrier, the pH may be pH 7 to 8, and preferably pH 7.2 to 7.4.

[0038] 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, and examples of the elution solution include an imidazole solution, an L-histidine solution, a buffer solution, an alkali metal salt solution, or water.

[0039] When an imidazole solution is used as the eluent, the concentration is preferably 0.005 to 1 M, and more preferably 0.05 to 0.1 M. Elution may be performed using an imidazole solution of a predetermined concentration, or, for example, elution may be performed using a concentration gradient of 0-100 mM imidazole.

[0040] When an L-histidine solution is used as the eluent, the concentration is preferably 0.005 to 1 M, and more preferably 0.05 to 0.1 M. Elution may be performed using an L-histidine solution of a predetermined concentration, or, for example, using a concentration gradient of 0 to 100 mM histidine.

[0041] When a buffer solution is used as the eluent, the pH can be appropriately adjusted depending on the type and concentration of the buffer solution used, but is preferably pH 6 to 10, more preferably pH 8.5 to 9. The concentration of the buffer solution can be 0.1 to 2 M, preferably 0.5 to 1 M.

[0042] The kit for purifying the polypeptide of the present invention may contain the above-mentioned expression vector of the present invention, and may also contain a carrier onto which one or more metal ions selected from nickel, copper, zinc, and cobalt are immobilized, and may also contain, as necessary, an instruction manual for purifying the polypeptide, and an adsorption solution, a washing solution, and an elution solution used in the above-mentioned method for purifying the polypeptide of the present invention.

[0043] The present invention will be described in more detail below with reference to examples. The examples are not limiting.

[0044] [Example 1] Although histidine tags consisting of six histidines have been widely used, it is known that the use of such tags with consecutive histidines reduces protein activity. Therefore, we investigated whether the reduction in protein activity could be suppressed by combining histidine with amino acids other than histidine. We produced red fluorescent protein (RFP protein) with a tag peptide consisting of consecutive histidines fused to the C-terminus of the RFP protein, and tag peptides with various amino acids arranged between three histidines. We created a strain that produces RFP fused with such tag peptides, and evaluated the activity of the protein by examining the fluorescence of RFP.

[0045] (a-1) 6H: HHHHHH (sequence number 2) (a-2) 12H: HHHHHHHHHHHH (sequence number 8) (a-3) 15H: HHHHHHHHHHHHHHHH (SEQ ID NO: 11) (a-4) 18H: HHHHHHHHHHHHHHHHHHH (sequence number 20) (a-5) 3HW×5: HHHWHHHWHHHWHHHWHHH (SEQ ID NO: 21) (a-6) 3HV × 5: HHHVHHHVHHHVHHHVHHH (SEQ ID NO: 22) (a-7) 3HT×5: HHHTHHHTHHHHTHHHHH (SEQ ID NO: 23) (a-8) 3HR × 5: HHHRHHHRHHHRHHHRHHH (SEQ ID NO: 24) (a-9) 3HM × 5: HHHMHHHMHHHHMHHHHH (sequence number 25) (a-10) 3HL×5: HHHLHHHLHHHLHHHLHHH (SEQ ID NO: 26) (a-11) 3HP × 5: HHHPHHHPHHHPHHHPHHH (SEQ ID NO: 27) (a-12) 3HN × 5: HHHNHHHHNHHHHNHHHH (SEQ ID NO: 28) (a-13) 3HK×5: HHHKHHHKHHHKHHHKHHH (SEQ ID NO: 29) (a-14) 3HQ × 5: HHHQHHHQHHHQHHHQHHH (sequence number 30) (a-15) 3HI×5: HHHIHHHIHHHIHHHHHH (SEQ ID NO: 31) (a-16) 3HF × 5: HHHFHHHFHHHFHHHFHHH (sequence number 32) (a-17) 3HE×5: HHHEHHHEHHHEHHHEHHHH (SEQ ID NO: 33) (a-18) 3HD×5: HHHDHHDHHHDHHHDHHHH (sequence number 34) (a-19) 3HC × 5: HHHCHHHCHHHWHHHCHHH (sequence number 35) (a-20) 3HY × 5: HHHYHHHYHHHYHHHYHHHH (SEQ ID NO: 36) (a-21) 3HS × 5: HHHSHHSHHSHHSHHSHHH (sequence number 37) (a-22) 3HG×5: HHHGHHHHHHHHHGHHH (sequence number 38) (a-23) 3HA × 5: HHHAHHAHHHAHHHAHHH (sequence number 39)

[0046] Using the YEpGAPcherry vector (see JP 2017-88536 A) as a template, primers were designed to add the above 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 each tag peptide.

[0047] Next, 1 mL of YPD was added to a 24-well plate, 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 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.

[0048] As shown in Figure 1, the more consecutive histidines there were, the lower the RFP activity was. In addition, it was revealed that the activity was 1.5 times higher when the tag had proline (P), glutamic acid (E), aspartic acid (D), glycine (G), alanine (A), asparagine (N), or glutamine (Q) between three consecutive histidines, compared to a tag with the same 15 histidines.

[0049] [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.

[0050] (b-1) No tag peptide (b-2) HHHHHH (sequence number 2) (b-3) HDHDHDHDHDH (SEQ ID NO: 16) (b-4) HHDHHDHH (SEQ ID NO: 40) (b-5) HHHDHHH (SEQ ID NO: 41) (b-6) HHHHHHDDDDDD (sequence number 42) (b-7) DDDDDDHHHHHH (SEQ ID NO: 43) (b-8) DDDHHHHHHDDD (SEQ ID NO: 44) (b-9) HHHHHHDDD (sequence number 45) (b-10) DDDHHHHHH (SEQ ID NO: 46)

[0051] Transformants were prepared that produced Gaussia luciferase (GLuc) fused with tag peptides (b-1) to (b-10) above, in which the tag peptides were added to the C-terminus of GLuc. The activity of the protein was evaluated by examining the fluorescence of GLuc.

[0052] Using a vector containing GLuc (New England Biolabs Japan) as a template, primers were designed to add the above tag peptide to the C-terminus of the GLuc protein, and PCR was performed to amplify the base sequence encoding the above tag peptide-fused GLuc by PCR. The PCR product was inserted into the above YEpGAPcherry vector by homologous recombination and transformed into the above BY4741 strain of Saccharomyces cerevisiae to produce transformants producing each tag peptide-fused GLuc.

[0053] Next, 1 mL of YPD was added to a 24-well plate, 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 OD600 was measured with 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.

[0054] As is clear from Figure 2, luciferase activity was more than twice as high when aspartic acid (D) was contained as compared with 6H, which has six consecutive histidines (H), and in particular, it was confirmed that activity was more than 10 times higher in HDHDHDHDHDH, in which histidines and aspartic acids are arranged alternately as shown in SEQ ID NO: 16, than in 6H. Therefore, it was considered that high luciferase activity could be maintained by reducing the number of consecutive histidines and / or the number of consecutive aspartic acids.

[0055] [Example 3] <Adsorption to nickel beads in a tube-1> It was confirmed from Example 2 that the use of HDHDHDHDHDH (6HD) consisting of the amino acid sequence shown in SEQ ID NO: 16 as a tag peptide suppresses the decrease in protein activity. Here, in order to purify a protein using a tag peptide, the tag peptide needs to be adsorbed to nickel. Thus, the adsorption of the tag peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 (HDHDHDHDHDH:6HD) or the tag peptide consisting of the amino acid sequence shown in SEQ ID NO: 17 (HEHEHEHEHEH:6HE) to nickel beads was examined.

[0056] (Preparation of RFP protein extract) As in Example 1 above, a transformant of the BY4741 strain of Saccharomyces cerevisiae was prepared using the YEpGAPcherry vector to produce a tag peptide fusion polypeptide (RFP-6HD) in which a tag peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 was 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 was fused to the C-terminus of the RFP protein. The transformant, the transformant producing RFP-6H prepared 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, stirred, and then centrifuged at 3000 rpm for 1 minute, and the supernatant was discarded. 1.5 mL of 0.1 M Tris base + 1% TritonX-100 was added thereto, stirred, and heated at 50°C for 1 hour. Thereafter, 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.

[0057] (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), mixed, and mixed at 30 rpm for 10 minutes using an Intelli-mixer, and then centrifuged at 6000 rpm for a few seconds. The results are shown in Figure 3.

[0058] 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>

[0059] (Effect of salt) In Example 3, Tris-HCl was used as the adsorption solution, but other adsorption solutions were used to examine the adsorption of the tag peptide fusion peptide to nickel beads. In Example 3, a protein extract produced in yeast was used as the tag peptide fusion peptide, but RFP-6HE and RFP-6H protein extracts produced in E. coli by the following method were used.

[0060] 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 an RFP-6HE or RFP-6H protein extract.

[0061] The adsorption solutions were 0.1M LiCl, 0.1M NaCl, and 0.1M KCl, and the concentrations of the adsorption solutions were 1M KCl, 0.1M KCl, and 0.01M KCl. The results for each adsorption solution are shown in Figure 4(a) and (b), respectively.

[0062] As is clear from FIGS. 4(a) and 4(b), it was revealed that RFP-6HE adsorbs to nickel beads in the presence of any of the salts 0.1 M LiCl, NaCl, and KCl. When distilled water was used as the adsorption solution, RFP-6HE did not adsorb to nickel beads (not shown). Also, in the case of KCl, there was no adsorption at a salt concentration of 0.01 M, but adsorption occurred at 0.1 M and 1 M. From these results, it was confirmed that RFP-6HE adsorbs to nickel beads in the presence of salts at a certain concentration or higher. On the other hand, RFP-6H adsorbed even in distilled water or 0.01 M KCl, and it was considered that RFP-6H adsorbs to nickel beads independently of the salt concentration. Usually, a KCl solution with a concentration of 1 M is the salt concentration used for elution in ordinary ion exchange chromatography. Therefore, if adsorption to nickel is carried out using KCl with a concentration of 1 M, it becomes possible to reduce non-specific adsorption of unwanted proteins.

[0063] Also, from the adsorption in the presence of a certain salt, it was suggested that the side chain of glutamic acid might be involved in the adsorption of RFP-6HE to nickel beads.

[0064] [Example 5] <Elution of RFP-6HE (Tube)> From Examples 3 and 4 above, it was confirmed that RFP-6HE adsorbs to nickel beads. Here, 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 could be eluted from the nickel beads.

[0065] (Elution with Imidazole) Using 2 M KPO4 pH 8.0 as the adsorption solution, 6HE was adsorbed to nickel beads in the same manner as in Example 3. Next, elution was performed with 0.1 M imidazole. The results are shown in FIG. 5. As is clear from FIG. 5, the entire tube turned bluish-purple, and it was confirmed that RFP-6HE elutes from nickel beads with 0.1 M imidazole in the same manner as RFP-6H.

[0066] (Elution with Histidine) Elution with imidazole was confirmed. However, since imidazole is basic, it may be necessary to remove imidazole from the eluate depending on the type of protein eluted and the use of the eluted protein. Here, imidazole is structurally partially similar to histidine. Also, histidine is lower in cost than imidazole and is easy to adjust in terms of pH. Therefore, elution with histidine (1, 10, 20, 40, 60, 80, 100 mM) was attempted 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 the nickel beads by histidine.

[0067] (Elution with Tris buffer) In elution using imidazole of the histidine tag, Tris buffer is often used to control the pH. Therefore, RFP-6HE was adsorbed onto nickel beads in the same manner as in Example 3 using 2M KPO4 pH 8.0 as the adsorption solution. Next, elution was performed with Tris buffer (pH 8, 9, and 10, concentrations 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 the nickel beads with any of the Tris buffers, and it was easier to elute with 1M than with 0.5M. In addition, it was clarified that while it was difficult to elute in the case of RFP-6H at 0.5M, RFP-6HE could be eluted, and it was particularly easy to elute at pH 9 and 10.

[0068] [Example 6]<Elution of RFP-6HE (Column)> In Example 5, elution was performed with a predetermined eluate using a tube, but in protein purification, it is common to perform elution using a column. Therefore, elution using a column was examined.

[0069] A nickel-packed 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-packed column, adsorbed with 1M KPO4 pH8.0, 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, and the absorption at UV280nm was measured. The eluted solution with a peak was analyzed by SDS-PAGE. The gel was electrophoresed at 300V for 35 minutes using Extra PAGE One Precast Gel 10-20%. The elution results with the concentration gradient of imidazole are shown in Figs. 8(a) and (b), and the results of SDS-PAGE analysis are shown in Fig. 8(c).

[0070] As is clear from Figs. 8(a) and (b), it was revealed that RFP-6HE eluted with a sharp peak and could be eluted with a low concentration of imidazole compared to RFP-6H. Also, as is clear from Fig. 8(c), it was confirmed that RFP-6HE was purified to the same extent as RFP-6H.

[0071] In Example 1, since the protein activity was high when proline (P) and glycine (G) were present between three consecutive histidines as a tag peptide, RFP-6HP and RFP-6HG were prepared in the same manner as in Example 2, and elution from the nickel-packed column was attempted with a concentration gradient of 0-100 mM imidazole as above. The results are shown in Figs. 9(a) and (b).

[0072] As is clear from Figs. 9(a) and (b), it was revealed that there was almost no elution from the nickel-packed column with RFP-6HP and RFP-6HG, and that RFP-6HE was more preferable than RFP-6HP and RFP-6HG for protein purification.

[0073] [Example 7]<Elution (Column) of RFP-6HE - 2> In Example 6, it was washed with distilled water before elution by the concentration gradient of imidazole. However, subsequent analysis by the present inventors confirmed that a part was eluted even with distilled water. Therefore, in order to further improve the purification degree, elution excluding the washing step with distilled water in the elution in Example 6 was examined. The results are shown in Fig. 10.

[0074] As is clear from Fig. 10, the elution of RFP-6HE became sharper compared to Fig. 8. Therefore, it became clear that it is better not to use water for washing before elution.

[0075] [Example 8]<Washing of RFP-6HE (column)> According to the results of Example 4, depending on the salt concentration, RFP-6HE is difficult to adsorb to nickel. On the other hand, in order to increase the purification degree, a lower salt concentration is preferable as a washing solution in order to prevent non-specific binding of unwanted proteins to nickel. Therefore, NaCl, KCl, LiCl, and RbCl were used to perform gradient washing, and the concentration was examined when washing with a salt solution.

[0076] Similar to Example 6, RFP-6HE was applied to a nickel-packed column. After adsorption with 1M KPO4 pH8.0, 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 of NaCl are shown in Fig. 11, and the results of KCl, LiCl, and RbCl are shown in Figs. 12(a)-(c).

[0077] As shown in Figs. 11 and 12, none of NaCl, KCl, LiCl, and RbCl were eluted at a concentration of 0.1M. On the other hand, at 0.01M, movement within the nickel column was observed. Therefore, from the viewpoints of improving the purification degree and the yield in protein purification, it was considered that 0.03M or more is appropriate as the concentration when washing with a salt solution.

[0078] [Example 9]<Elution of RFP-6HE (Column)-3> In Example 8, when washing with NaCl, KCl, LiCl, and RbCl, it was confirmed that RFP-6HE remained adsorbed to the nickel column if the concentration was around 0.03M. Therefore, after the above washing, it was examined whether elution was possible with imidazole, histidine, or water.

[0079] Similar to Example 6, RFP-6HE was applied to a nickel-packed column. After adsorption with 1M KPO4 pH 8.0, washing was performed with 0.05M KCl for 30 minutes, and then elution was carried out with imidazole or L-histidine having a concentration gradient of 0 - 100 mM. The results are shown in FIGS. 13 and 14.

[0080] As is clear from FIG. 13, sharp peaks were observed with imidazole at low concentrations (5 - 20 mM). Also, as is clear from FIG. 14, sharp elution occurred with L-histidine at low concentrations (5 - 15 mM).

[0081] Furthermore, similar to Example 6, RFP-6HE was applied to a nickel-packed column. After adsorption with 1M KPO4 pH 8.0, washing was performed with 0.1M KCl for 50 minutes, and then elution was carried out with distilled water for about 30 minutes. The results are shown in FIG. 15.

[0082] As is clear from FIG. 15, it became clear that sharp elution also occurred with water after washing with a low concentration of salt.

[0083] [Example 10]<Elution of DNA Polymerase (Column)> In the above, purification of RFP was performed, but purification with DNA polymerase as another protein was examined.

[0084] A transformant of Escherichia coli HST08 strain producing a tag peptide fused DNA polymerase in which the tag peptide 6HE was 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% TritonX-100 was added and stirred, followed by heating at 95°C for 5 minutes.

[0085] The DNA polymerase-6HE was applied to a nickel-packed column in the same manner as in Example 6, and after adsorption with 1M KPO4 pH 8.0, it was washed with 1M KCl for 30 minutes and eluted with 100 mM imidazole for about 30 minutes, and UV280nm was measured. The results are shown in Figure 16.

[0086] As is clear from Figure 16, it was confirmed that DNA polymerase-6HE was eluted very sharply. Therefore, it was demonstrated that the use of the present tag peptide makes it possible to purify proteins to high purity regardless of the type of protein.

Claims

1. A tag peptide consisting of any one of the following polypeptides (1-3') to (1-9'): (1-3') Two or three histidines in the amino acid sequence shown in SEQ ID NO:3 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 residues, or three or four aspartic acid-histidine or glutamic acid-histidine residues; (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;

2. 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.

3. 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.

4. A tag peptide fusion polypeptide in which the tag peptide according to any one of claims 1 to 3 is fused with a polypeptide.

5. The tag peptide fusion polypeptide according to claim 4, wherein the polypeptide fused to the tag peptide is a protein.

6. A polynucleotide encoding the tag peptide according to any one of claims 1 to 5.

7. An expression vector containing the polynucleotide of claim 6.

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 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. 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. 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.

11. 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.

12. The method for purifying a polypeptide according to any one of claims 8 to 11, wherein the metal ion is nickel.

13. A kit for purifying a polypeptide, comprising the expression vector according to claim 7.

14. (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 units, or four aspartic acid-histidine or glutamic acid-histidine units; or (1-5″) Four histidines in the amino acid sequence shown in SEQ ID NO:5 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.

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