JP2017037014A - Binder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a binder with metal-biding capacity and metal-binding proteins coupled with such a binder in view of the fact that there is a need for a versatile method of immobilizing proteins on electrodes that allows for controlling orientation of immobilization.SOLUTION: Inventors have found a peptide with metal-binding capacity. The inventors further found that coupling the peptide to a protein allows the protein to be bound to metal, which lead to completion of the present invention.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a binder having a metal binding ability.

  An electrode in which a protein such as an antibody or an enzyme is immobilized on the surface of the electrode is used for a biosensor or a biobattery. For immobilization, a method using a crosslinking agent is known, but there is a problem that it is difficult to control protein orientation (Patent Document 1).

  An immobilization method for controlling the orientation is also known, but the electrode constituent base material is limited to the carbon base material, or the enzyme needs to have a specific structure, and there is a problem that versatility is low ( Patent Document 2 or 3).

  On the other hand, some metal binding peptides have been known (Non-patent Documents 1 and 2).

JP 2014-6154 A JP 2005-83873 A Japanese Patent No. 5219265

“Nature Materials” (UK), 2003, Volume 2, p. 577-585 “Acta Biomaterialia”, (USA), 2007, Vol. 3, No. 3, p. 289-299

  There has been a demand for a versatile immobilization method that can control the orientation in immobilizing proteins to electrodes. The present invention has been made in view of the above, and provides a binding agent having metal binding ability and a metal binding protein to which the binding agent is linked.

  The inventors have found a peptide having a metal binding ability. Furthermore, the inventors have found that a protein can be bound to a metal by linking the peptide to a protein, and the present invention has been completed.

That is, the present invention relates to the following aspects [1] to [14].
[1] A binder comprising a metal-binding peptide having the following properties (i), (ii), (iii) and (iv):
(I) has gold and platinum group metal binding ability;
(Ii) is a basic peptide;
(Iii) is a hydrophilic peptide;
(Iv) It has an orientation control ability.
[2] The binder according to [1], comprising a metal-binding peptide in which the total ratio of R and K in the whole peptide is at least 30%.
[3] The binding agent according to [1] or [2], comprising a metal-binding peptide of 6 amino acids.
[4] The binder according to any one of [1] to [3], which has the following properties (v), (vi) and (vii):
(V) the isoelectric point is 8.5 to 13.5;
(Vi) the hydrophilicity / hydrophobicity index is -0.200 to -4,000;
(Vii) It consists of 4 to 14 amino acids.
[5] A binding agent comprising a peptide having the following amino acid sequence (a) or (b) and having a binding ability to metal:
(A) the amino acid sequence according to any one of (1) to (17) below:
(1) an amino acid sequence consisting of K, K, R, E, V and R;
(2) an amino acid sequence consisting of V, Y, N, K, R and K;
(3) an amino acid sequence consisting of S, R, A, A, K and Y,
(4) an amino acid sequence consisting of Q, K, R, K, V and V;
(5) an amino acid sequence consisting of K, G, R, G, R and V;
(6) an amino acid sequence consisting of K, R, K, A, A and M;
(7) an amino acid sequence consisting of K, T, R, G, V and K;
(8) an amino acid sequence consisting of K, Q, K, K, T and T;
(9) an amino acid sequence consisting of R, T, R, N, R and S,
(10) an amino acid sequence consisting of T, Q, K, G, R and K;
(11) an amino acid sequence consisting of K, G, A, K, K and V,
(12) an amino acid sequence consisting of K, K, T, S, K and G;
(13) an amino acid sequence consisting of K, T, R, M, R and G;
(14) an amino acid sequence consisting of L, K, D, K, K and K;
(15) an amino acid sequence consisting of R, G, Y, K, K and G;
(16) an amino acid sequence consisting of R, K, G, N, K and A,
(17) An amino acid sequence consisting of R, V, G, R, K and G, wherein one or several amino acids are deleted, substituted or added in the amino acid sequence described in (b) (a).
[6] The binding agent according to [5], comprising a peptide having the following amino acid sequence (c), (d) or (e) and having a binding ability to a metal:
(C) the amino acid sequence according to any one of SEQ ID NOs: 1 to 17;
(D) a reverse sequence of the amino acid sequence described in (c);
(E) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in (c) or (d).
[7] The method according to any one of [1] to [6], comprising an amino acid selected from K, R, G, V, T, A, Q, S, Y, N, M, L, D, or E. Binder.
[8] A metal-binding protein in which the binding agent according to any one of [1] to [7] is linked to a protein.
[9] The metal binding protein according to [8], wherein a binder is linked to the N-terminus and / or C-terminus of the protein.
[10] The metal binding protein according to [8] or [9], wherein the protein is an antibody or an enzyme.
[11] The metal binding protein according to any one of [8] to [10], wherein the protein is an oxidoreductase.
[12] A measurement reagent composition comprising the metal binding protein according to any one of [8] to [11].
[13] A method for measuring an object to be measured in a sample, which uses the metal binding protein according to any one of [8] to [11].
[14] The measurement method according to [13], wherein the measurement target is glucose.

  According to the present invention, a binding agent comprising a peptide having a metal binding ability can be used. Furthermore, a metal binding protein can be obtained by linking the binder to a protein such as an antibody or an enzyme. Since the protein easily binds to a metal, the protein can be easily fixed to the electrode and can be fixed with the orientation of the protein.

It is a schematic diagram of a metal binding protein fixed electrode. From the left, the protein N-terminal, C-terminal, and immobilization of a metal-binding protein in which a metal-binding peptide is linked to the N-terminal and C-terminal are shown. It is a schematic diagram of metal binding esterases Est-N1-17 and Est-C1-17. It is an evaluation result of the binding ability of metal binding esterase. It is an orientation control ability evaluation result of metal binding o-acetylserine sulfhydrylase. It is a schematic diagram of metal binding glucose dehydrogenase GLD-N1, GLD-C1 'and GLD-N1C1'. It is an evaluation result of the binding ability of metal binding glucose dehydrogenase GLD-N1C1 '.

The binder of the present invention comprises a metal-binding peptide having the following characteristics.
(I) Gold and platinum group metal binding peptides.
(Ii) It is a basic peptide.
(Iii) It is a hydrophilic peptide.
(Iv) It has an orientation control ability.

The binder of the present invention preferably comprises a metal-binding peptide having the following characteristics.
(V) The isoelectric point is 8.5 to 13.5, preferably 9.0 to 13.0, and more preferably 9.5 to 12.5.
The isoelectric point is a theoretical isoelectric point (Theoretical pI) calculated by the ExPASy ProtParam tool (http://web.expasy.org/protparam/).
(Vi) The hydrophilicity / hydrophobicity index is -0.200 to -4,000, preferably -0.500 to -3.500, and more preferably -0.800 to -3.200.
In addition, the hydrophilicity / hydrophobicity index is a hydrophilicity / hydrophobicity index (GRAVY) calculated by ExPASy's ProtParam tool (http://web.expasy.org/protparam/).
(Vii) A binding agent consisting of 4 to 14 amino acids, preferably 5 to 13 amino acids or 6 to 12 amino acids, more preferably 10 amino acids, 9 amino acids, 8 amino acids or 7 amino acids. More preferably, it consists of 6 amino acids.

  The binder of the present invention is preferably (viii) a metal-binding peptide in which the total proportion of arginine (R) and lysine (K) in the total peptide is at least 20%. More preferably, the total ratio of R and K in the whole peptide is at least 25%, 30%, 35% or 40%.

The binding agent of the present invention preferably comprises a peptide having the amino acid sequence (a) or (b) and has a binding ability to metal.
(A) The amino acid sequence according to any one of (1) to (17) below.
(1) An amino acid sequence consisting of K, K, R, E, V and R.
(2) An amino acid sequence consisting of V, Y, N, K, R and K.
(3) An amino acid sequence consisting of S, R, A, A, K and Y.
(4) An amino acid sequence consisting of Q, K, R, K, V and V.
(5) An amino acid sequence consisting of K, G, R, G, R and V.
(6) An amino acid sequence consisting of K, R, K, A, A and M.
(7) An amino acid sequence consisting of K, T, R, G, V and K.
(8) An amino acid sequence consisting of K, Q, K, K, T, and T.
(9) An amino acid sequence consisting of R, T, R, N, R and S.
(10) An amino acid sequence consisting of T, Q, K, G, R and K.
(11) An amino acid sequence consisting of K, G, A, K, K and V.
(12) An amino acid sequence consisting of K, K, T, S, K and G.
(13) An amino acid sequence consisting of K, T, R, M, R and G.
(14) An amino acid sequence consisting of L, K, D, K, K and K.
(15) An amino acid sequence consisting of R, G, Y, K, K and G.
(16) An amino acid sequence consisting of R, K, G, N, K and A.
(17) An amino acid sequence consisting of R, V, G, R, K and G.
The order of amino acids constituting any of the amino acid sequences described in (1) to (17) is not limited. More preferably, it is a sequence consisting of the order described above, or its reverse sequence. More preferably, it consists of a peptide consisting of the amino acid sequence described in any one of (1) to (17) and has the ability to bind to a metal. Preferably, it has at least one characteristic of (i) to (viii). In addition, all the amino acids described in (1) to (17) are represented by one letter.
(B) It consists of a peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in (a) above, and has a binding ability to metal. The number is preferably 3, and more preferably 2.

The binding agent of the present invention is preferably composed of a peptide having the following amino acid sequence (c), (d) or (e) and has a binding ability to a metal. Preferably, it has at least one characteristic of (i) to (viii).
(C) The amino acid sequence according to any one of SEQ ID NOs: 1 to 17.
(D) A reverse sequence of the amino acid sequence described in (c).
(E) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in (c) or (d). The number is preferably 3, and more preferably 2.

The binding agent of the present invention is preferably composed of a peptide having the following amino acid sequence (f) and has a binding ability to metal.
(F) An amino acid sequence in which two sequences are tandemly linked among the amino acid sequences described in (a), (b), (c), (d) or (e). The combination of two sequences that are tandemly linked is not particularly limited as long as it is an amino acid sequence described in (a), (b), (c), (d), or (e), and may be a combination of the same sequences.

  The binder of the present invention preferably consists of an amino acid selected from K, R, G, V, T, A, Q, S, Y, N, M, L, D or E. Preferably, it does not contain at least one amino acid selected from C, H, P, W, I or F.

  The binding agent of the present invention is not particularly limited as long as it is a metal-binding peptide having the above sequence, and may be a peptide obtained by gene recombination or a peptide obtained by synthesis. The gene recombination may be performed by a well-known method using the base sequence encoding the amino acid sequence described in (a), (b), (c), (d), (e) or (f). .

  The binding agent of the present invention may be a peptide having a metal binding ability, and may be any peptide that can chemically bind to a metal. The metal to which the binder can bind is preferably gold or a platinum group metal. For example, platinum group metals can be exemplified by platinum, palladium, rhodium, ruthenium or iridium, with palladium being more preferred. The binding agent of the present invention can bind to an electrode using the metal as a conductive substance, and can immobilize an enzyme or antibody used for measurement on the electrode.

  A metal-binding protein can be produced by linking the binder to a protein. The binding agent is preferably linked to the N-terminus and / or C-terminus of the protein. When linking a binding agent to the N-terminal of the protein, it is preferable to link it to the N-terminal of a mature protein that does not contain a secretory signal sequence. Furthermore, there may be a linker peptide between the protein and the binder. The linking method may be total synthesis of the entire metal binding protein or may be a genetic engineering technique. If it is a binder of this invention, even if it connects with a protein, it will hardly exert a bad influence on the structure of a protein, and can maintain the characteristic of a protein.

  For example, in the genetic engineering technique, a vector into which a base sequence encoding a metal binding protein is inserted is prepared. Finally, a host microorganism is transformed using the expression vector to obtain a transformant. Subsequently, the transformant is cultured, and a metal binding protein can be obtained from the obtained culture.

  The vector is a cloning vector or an expression vector, and includes the base sequence as an insert. As the transformant, for example, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as fungi (yeast, Aspergillus genus Ascomycetes, basidiomycetes, etc.), insect cells, mammalian cells and the like are used. Can do. The vector may be maintained in a transformed cell in the state of a plasmid, or may be maintained by being incorporated into a chromosome. Furthermore, the host can be appropriately selected according to the necessity, such as the necessity of sugar chains, unnecessary, secretory production, and the like. When a metal binding protein in which a binder is linked to the N-terminal side of the protein is secreted to the host, the base sequence is designed so that a secretory signal sequence exists before the binding agent sequence. Alternatively, a secretory signal sequence on the vector side may be used. When a metal-binding protein having a binding agent linked to the C-terminal side of the protein is produced in the host, a gene sequence encoding the binding agent is inserted before the stop codon of the protein gene. When a linker peptide is inserted between the protein and the binding agent, a base sequence encoding the linker peptide may be inserted between each gene.

  The protein linking the binder may be a glycoprotein. The protein is preferably an enzyme or an antibody. When the binding agent is linked to them, a metal-binding enzyme or a metal-binding antibody is obtained. Examples of the protein include hydrolase, transferase, and oxidoreductase. When the binder is linked to them, metal-bonded hydrolase, metal-bound transferase, or metal-bound oxidoreductase is obtained. For example, the hydrolase can be exemplified by esterase, and the transferase can be exemplified by o-acetylserine sulfhydrylase. The oxidoreductase includes an oxidase and a dehydrogenase. Examples thereof include enzymes that catalyze an oxidation reaction using glucose, lactic acid, cholesterol, or alcohol as substrates. Glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, alcohol oxidase or alcohol dehydrogenase are preferred.

  As the protein, a protein having a known sequence may be used. For example, a protein having the amino acid sequence described in SEQ ID NOs: 18 to 22 can be used. A metal-binding protein is produced by linking the binding agent sequence described in any of (a) to (f) above to the N-terminal and / or C-terminal of the amino acid sequence described in SEQ ID NOs: 18 to 22. Also good. An amino acid sequence having at least 80%, 85%, 90%, 95% or 98% similarity to any of the amino acid sequences set forth in SEQ ID NOs: 18-22 may be used. Any of the amino acid sequences described in SEQ ID NOs: 18 to 21 may be used in which one or more amino acids are deleted, substituted, or added. The number of mutations is preferably at most 60, 55, 50, 40, 30, 20, 20, 15, 5, 3, or 2. When the base sequence encoding these amino acid sequences is used, the metal binding protein of the present invention can be produced by the genetic engineering technique.

  A metal-binding protein-immobilized electrode can be produced by immobilizing the metal-binding protein of the present invention on the electrode by chemical bonding. The metal-binding protein of the present invention can be easily bound to an electrode provided with the metal conductive material via a binder portion, and can be immobilized with orientation. A schematic diagram of the metal-binding protein-immobilized electrode is shown in FIG.

  A biosensor including an electrode on which the metal binding protein of the present invention is immobilized and an insulating substrate can be produced. For example, an electrochemical biosensor can be manufactured by including a step of forming an electrode system using a method such as screen printing or vapor deposition on an insulating substrate, and a step of immobilizing a protein. The electrode preferably includes a counter electrode and a working electrode, and may include a reference electrode. It is possible to construct a biosensor that detects a current value, ions, color intensity, or frequency change. In addition, mediators, buffers or stabilizers may be included.

  Examples of the mediator include proteinaceous electron mediators such as heme, ferricyanide compounds, quinone compounds, osmium compounds, phenazine compounds, and phenothiazine compounds. The buffer may be any buffer that can be adjusted to the target reaction environment. Examples of stabilizers include bovine serum albumin (BSA) or ovalbumin, saccharides or sugar alcohols having no activity with molecular identification elements, carboxyl group-containing compounds, alkaline earth metal compounds, ammonium salts, sulfates or proteins. It can be illustrated.

  By using the biosensor of the present invention, a measurement method for measuring a measurement object in a sample solution can be provided. By providing the step of bringing the biosensor into contact with the sample solution, the measurement object in the sample solution can be measured. The protein to be immobilized may be appropriately selected depending on the desired measurement object. For example, when measuring glucose, a glucose measuring method can be provided by immobilizing glucose oxidase or glucose dehydrogenase on an electrode. The sample solution is not particularly limited, but a biological sample can be exemplified, and specific examples include blood, tears, urine, cell interstitial fluid, and the like. For blood glucose measurement, eukaryotic cell-derived glucose dehydrogenase using flavin adenine dinucleotide as a coenzyme is preferable.

[Example 1]
(Acquisition of metal-binding peptide)
In order to obtain a metal-binding peptide having metal-binding ability, a random peptide library consisting of 6 amino acid sequences was constructed by the phage display method, and biopanning was performed. Specifically, cycles (1) to (3) or (4) were performed under the following cleaning conditions I, II, or III. Finally, 17 types of phages that bind tightly to the palladium (Pd) plate were obtained and subjected to DNA sequence analysis. Table 1 shows the amino acid sequences of 17 metal-binding peptides (P1 to P17) determined from the analyzed DNA sequences. For each amino acid sequence, the theoretical isoelectric point (Theoretical pI) and the hydrophilicity / hydrophobicity index (GRAVY) of ExPASy's ProtParam tool (http://web.expasy.org/protparam/) Calculated and listed in Table 1. All were basic peptides. The Au, Pt or Pd binding peptides described in Non-Patent Documents 1 and 2 are summarized in Table 2.

Cleaning condition I
Cycle (1): Step 1 → Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5 →
Cycle (2): Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5 →
Cycle (3): Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5
Cleaning condition II
Cycle (1): Step 1 → Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5 →
Cycle (2): Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5 →
Cycle (3): Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5 →
Cycle (4): Step 2 → Step 3 (FBS × 1 time, PBS × 2 times) → Step 4 → Step 5
Cleaning condition III
Cycle (1): Step 1 → Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5 →
Cycle (2): Step 2 → Step 3 (1% SDS × 1 time, PBS × 2 times) → Step 4 → Step 5 →
Cycle (3): Step 2 → Step 3 (PBS × 3 times) → Step 4 → Step 5 →
Cycle (4): Step 2 → Step 3 (FBS × 1 time, PBS × 2 times) → Step 4 → Step 5
* PBS: phosphate buffered saline, FBS: fetal bovine serum

Step 1: Prepare a T7 phage suspension displaying random peptides.
Step 2: The Pd plate is immersed in a T7 phage suspension at 37 ° C. for 1 hour to bind the T7 phage to the Pd plate.
Step 3 (Washing Step): The Pd plate to which T7 phage is bound is immersed in a buffer solution of each washing condition at 37 ° C. for 5 minutes for washing. The washing is performed for each washing condition.
Step 4: The washed Pd plate is immersed in a culture solution of E. coli BL21 strain, and T7 phage remaining on the Pd plate is infected with E. coli BL21 for amplification.
Step 5: Confirm amplification of T7 phage by titer check.

[Example 2]
(Metal-binding esterase)
(1) Preparation of metal-binding esterase Binding peptides P1 to P17 having the amino acid sequences described in SEQ ID NOs: 1 to 17 were linked to the N-terminus or C-terminus of esterase (Est) described in SEQ ID NO: 18, respectively. Types of metal binding Est were made. Each metal binding Est is, respectively, Est-N1, Est-N2, Est-N3, Est-N4, Est-N5, Est-N6, Est-N7, Est-N8, Est-N9, Est-N10, Est. -N11, Est-N12, Est-N13, Est-N14, Est-N15, Est-N16, Est-N17, Est-C1, Est-C2, Est-C3, Est-C4, Est-C5, Est-C6 Est-C7, Est-C8, Est-C9, Est-C10, Est-C11, Est-C12, Est-C13, Est-C14, Est-C15, Est-C16 or Est-C17.

  All metal binding Ests were produced by genetic engineering techniques. As the vector, a His tag fusion expression vector into which a gene expressing Est described in SEQ ID NO: 18 was inserted was used.

  In order to express Est-N1, a base sequence encoding the amino acid sequence described in SEQ ID NO: 1 is inserted before the start codon of the Est gene in the vector, and an expression vector pEst-N1 containing the entire length of the Est-N1 gene is inserted. Produced. Similarly, for Est-N2-17, expression vectors pEst-N2-17 containing the entire length of the Est-N2-17 gene were prepared.

  An expression vector pEst-C1 containing the entire length of the Est-C1 gene is inserted by inserting a base sequence encoding the amino acid sequence described in SEQ ID NO: 1 before the stop codon of the Est gene in the vector so that Est-C1 is expressed. Produced. Similarly, for Est-C2-17, expression vectors pEst-C2-17 containing the entire length of the Est-C2-17 gene were prepared.

  Escherichia coli was transformed with each of the vectors to produce a total of 34 types of recombinant Escherichia coli producing Est-N1-17 or Est-C1-17. Each recombinant strain was cultured in a liquid medium containing ampicillin and collected. Each recombinant E. coli collected was sonicated and each cell-free extract (CFE) containing Est-N1-17 or Est-C1-17 was recovered. Each CFE was heat-treated and then purified with a His tag fusion protein purification column, and purified Est-N1-17 or purified Est-C1-17 total 34 samples were collected. About each purified enzyme, when confirmed by SDS-PAGE, the contaminating protein was able to be removed. Furthermore, any metal-binding Est had the same activity as Est without linking a metal-binding peptide. It was found that even when the metal-binding peptide of the present invention was linked to the N terminus and / or C terminus of Est, inactivation due to ligation was not observed, and enzyme activity could be retained. A schematic diagram of each metal binding property Est is shown in FIG.

(2) Binding ability evaluation of metal binding esterase The binding ability to palladium was evaluated about each of 34 types of metal binding Est obtained in (1) of Example 2. A 1 cm × 1 cm plastic plate (Pd plate), one side of which was plated with palladium, was fixed to a 24-hole plate with a double-sided tape with the plating side facing up. 800 μL of 10 μg / ml Est-N1 solution was added to the plate and allowed to stand at 37 ° C. for 1 hour to bind Est-N1 on the Pd plate. Next, a washing step of immersing the Pd plate in PBS containing 0.1% Tween 20 at 37 ° C. for 5 minutes was performed three times. Furthermore, it was immersed in PBS at 37 ° C. for 5 minutes to remove unbound Est-N1.

  In order to measure the Est activity on the Pd plate, the Pd plate was transferred to a 5 mL tube containing 4.95 mL PBS that had been kept at 37 ° C. in advance. 50 μL of 100 mM p-nitrophenylacetic acid was added to the tube to start the enzyme reaction. 100 μL was sampled every 5 minutes and placed in a 96-well plate, and the absorbance at 405 nm was measured with a plate reader. Est-N2-17 and Est-C-17 were also measured in the same manner as described above. In addition, it measured similarly to the above using Est which has not connected metal binding peptide as control. The results are shown in FIG. On the horizontal axis, metal-binding peptides P1 to P17 linked to the N-terminus or C-terminus are represented by P1 to P17, and the control is represented by C. On the vertical axis, the residual activity of esterase on the Pd plate is expressed as a relative value with the control being 1.

  As a result, Est-N1-17 and Est-C1-17 had about 1.5 to 4.0 times the residual activity of Est on the Pd plate as compared with Est not linked with the metal-binding peptide. . Therefore, any of the metal-binding esterases can increase the metal-binding ability by the linked metal-binding peptide, can be appropriately bound on the Pd plate, and can exhibit high residual activity on the Pd plate. It seems to have become.

[Example 3]
(Metal-binding o-acetylserine sulfhydrylase)
(1) Production of metal-binding o-acetylserine sulfhydrylase Metal-binding peptide P1, 2, 3, 4, 9 or 13 having the amino acid sequence described in SEQ ID NO: 1, 2, 3, 4, 9 or 13 12 types of metal-binding OASS were prepared by linking each to the N-terminal or C-terminal of o-acetylserine sulfhydrylase (OASS) described in SEQ ID NO: 19. Each metal-bonded OASS is OASS-N1, OASS-N2, OASS-N3, OASS-N4, OASS-N9, OASS-N13, OASS-C1, OASS-C2, OASS-C3, OASS-C4, OASS, respectively. -C9 or OASS-C13.

  All metal-binding OASS was produced by a genetic engineering technique. As the vector, a His tag fusion expression vector into which a gene expressing OASS described in SEQ ID NO: 19 was inserted was used.

  An expression vector pOASS-N1 containing the full length of the OASS-N1 gene is inserted by inserting a base sequence encoding the amino acid sequence described in SEQ ID NO: 1 before the start codon of the OASS gene in the vector so that OASS-N1 is expressed. Produced. Similarly, expression vectors pOASS-N3, pOASS-N4, pOASS-N9 and pOASS-N13 were prepared for OASS-N2, OASS-N3, OASS-N4, OASS-N9 and OASS-N13.

  An expression vector pOASS-C1 containing the full length of the OASS-C1 gene is inserted by inserting a base sequence encoding the amino acid sequence described in SEQ ID NO: 1 before the stop codon of the OASS gene in the vector so that OASS-C1 is expressed. Produced. Similarly, for OASS-C2, OASS-C3, OASS-C4, OASS-C9 and OASS-C13, expression vectors pOASS-C2, pOASS-C3, pOASS-C4, pOASS-C9 and pOASS-C13 were prepared.

  E. coli was transformed with each of the vectors, and OASS-N1, OASS-N2, OASS-N3, OASS-N4, OASS-N9, OASS-N13, OASS-C1, OASS-C2, OASS-C3, OASS-C4 Twelve kinds of recombinant Escherichia coli producing OASS-C9 or OASS-C13 were prepared. Each recombinant strain was cultured in a liquid medium containing ampicillin and collected. Each recombinant E. coli collected was sonicated and OASS-N1, OASS-N2, OASS-N3, OASS-N4, OASS-N9, OASS-N13, OASS-C1, OASS-C2, OASS-C3, OASS Each cell-free extract (CFE) containing -C4, OASS-C9 or OASS-C13 was collected. Then, purified with a His tag fusion protein purification column, purified OASS-N1, OASS-N2, OASS-N3, OASS-N4, OASS-N9, OASS-N13, OASS-C1, OASS-C2, OASS-C3, A total of 12 samples of OASS-C4, OASS-C9 or OASS-C13 were collected. About each purified enzyme, when confirmed by SDS-PAGE, the contaminating protein was able to be removed.

  In addition, the metal binding OASS which connected palladium binding peptide: SPHPGPY of nonpatent literature 1 to the N terminal or C terminal of OASS was created similarly to the above, and it was set as control.

(2) Evaluation of ability to control orientation of metal binding o-acetylserine sulfhydrylase About each of 12 types of metal binding OASS obtained in (1) of Example 3, NAPiCOS system (manufactured by Nippon Denpa Kogyo Co., Ltd.) Was used to evaluate the orientation control ability. First, for a QCM sensor (manufactured by Nippon Denpa Kogyo Co., Ltd.), a palladium film-forming QCM sensor was produced by performing palladium vapor deposition on the gold electrode surface of the crystal resonator. On the palladium of the sensor, a 100 μg / mL OASS-N1 solution was added and allowed to stand at room temperature for 1 hour to bind OASS-N1 to the palladium surface. Next, the sensor was washed with MilliQ water to remove unbound OASS-N1, and an OASS-N1 immobilized sensor chip was produced. The sensor was set in a NAPiCOS system and blocked with a 1 mg / mL BSA solution. A 10 μg / mL serine acetyltransferase (SAT) solution was flowed, and then a 100 μg / mL SAT solution was flowed to interact with OASS-N1 on the sensor, and each frequency change was measured. Similar operations for OASS-N2, OASS-N3, OASS-N4, OASS-N9, OASS-N13, OASS-C1, OASS-C2, OASS-C3, OASS-C4, OASS-C9, OASS-C13 or control The frequency change was measured. The results are shown in FIG. In FIG. 4, the metal-binding peptide P1, P2, P3, P4, P9 or P13 linked to the N-terminus or C-terminus is represented by P1, P2, P3, P4, P9 or P13, and the control is PC. expressed. The dotted line represents the change in the frequency of the metal-binding OASS having the metal-binding peptide linked to the N-terminal side, and the solid line represents the frequency of the metal-binding OASS having the metal-binding peptide linked to the C-terminal side. It represents a change.

  When a metal-binding peptide is linked to the N-terminal side of the protein, it binds to the metal at the N-terminal side, while when a metal-binding peptide is linked to the C-terminal side, it binds to the metal at the C-terminal side. Since OASS has an active site near the C-terminal, theoretically, SAT hardly acts when bound to a metal on the C-terminal side, so the measured value is small. When bound to metal on the N-terminal side, SAT acts on the active site. It is thought that the measured value becomes large because it is easy to do. Therefore, comparing the measured values of OASS-N1 and OASS-C1, if OASS-N1 has a larger frequency change, it can be determined that orientation control is performed by the metal-binding peptide.

  From FIG. 4, OASS-N1 and OASS-C1 (P1), OASS-N2 and OASS-C2 (P2), OASS-N3 and OASS-C3 (P3), OASS-N4 and OASS-C4 (P4), OASS- N9 and OASS-C9 (P9) or OASS-N13 and OASS-C13 (P13) all had a greater frequency change when the metal-binding peptide was linked to the N-terminus. On the other hand, in the control (PC), there was no difference between the N-terminal side and the C-terminal side in which metal-binding peptides were linked. That is, it was clarified that the orientation was hardly controlled in the control, and the orientation was controlled in the metal-binding peptide of the present invention.

[Example 4]
(Metal-binding glucose dehydrogenase)
(1) Preparation of metal-binding glucose dehydrogenase Three types of metal-binding glucose dehydrogenase were prepared by connecting binding peptides having the amino acid sequence described in SEQ ID NO: 1 or its reverse sequence. The first is GLD-N1 in which a binding peptide having the amino acid sequence described in SEQ ID NO: 1 is linked to the N-terminus of glucose dehydrogenase (GLD). The second is GLD-C1 ′ in which a binding peptide having the reverse sequence of the amino acid sequence described in SEQ ID NO: 1 is linked to the C-terminal of GLD. The third is GLD-N1C1 ′ in which a binding peptide having the amino acid sequence described in SEQ ID NO: 1 is connected to the N-terminal of GLD, and a binding peptide having the reverse sequence of the amino acid sequence described in SEQ ID NO: 1 is connected to the C-terminal. is there. GLD-N1, GLD-C1 ′ and GLD-N1C1 ′ were all produced by genetic engineering techniques. As the vector, an expression vector into which a gene expressing GLD described in SEQ ID NO: 20 was inserted was used. This vector is a vector in which the signal sequence gene portion of the vector described in Example 4 of WO 2006/101239 is replaced with a gene encoding MLFSLAFLSALSLATASPAGRA.

  A base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 was inserted after the signal gene sequence in the vector so that GLD-N1 was expressed, to prepare an expression vector pGLD-N1 containing the full length of the GLD-N1 gene. .

  In order to express GLD-C1 ′, a base sequence encoding the reverse sequence of the amino acid sequence described in SEQ ID NO: 1 is inserted before the stop codon of the GLD gene in the vector, and the entire length of GLD-C1 ′ gene is included. An expression vector pGLD-C1 ′ was prepared.

  In order to express GLD-N1C1 ′, a base sequence encoding the reverse sequence of the amino acid sequence described in SEQ ID NO: 1 is inserted in front of the stop codon of the GLD gene in pGLD-N1, and the full length of the GLD-N1C1 ′ gene An expression vector pGLD-N1C1 ′ containing

  Aspergillus oryzae NS4 strain is transformed with each of the vectors by the method described in Example 4 of WO2006 / 101239 pamphlet, and GLD-N1, GLD-C1 ′ or GLD-N1C1′-producing recombinant Aspergillus oryzae 3 A seed was produced. Each recombinant strain was cultured in a liquid medium, and each culture supernatant was collected. Each culture supernatant was purified by an anion exchange column and confirmed by SDS-PAGE. As a result, single band GLD-N1, GLD-C1 'or GLD-N1C1' could be confirmed. Furthermore, any metal-binding GLD had the same activity as GLD not linked with a metal-binding peptide. It was found that even when the metal-binding peptide of the present invention was linked to the N-terminus and / or C-terminus of GLD, inactivation due to the linkage was not observed and the enzyme activity could be retained. A schematic diagram of each metal-binding GLD is shown in FIG.

(2) Evaluation of metal binding glucose dehydrogenase GLD-N1C1 ′ obtained in (1) of Example 4 was evaluated for its ability to bind to palladium. A 1 cm × 1 cm plastic plate (Pd plate) with one side plated with palladium was fixed to a 12-hole plate with a double-sided tape with the plated side facing up. 1 mL of a 7 μg / ml GLD-N1C1 ′ solution was added to the plate and left at 37 ° C. for 1 hour to bind GLD-N1C1 ′ on a Pd plate. Next, a washing step of immersing the Pd plate in 5 mL of PBS containing 0.05% Tween 20 at room temperature for 5 minutes was performed three times. Furthermore, it was immersed in PBS for 5 minutes at room temperature to remove unbound GLD-N1C1 ′.

  In order to measure the GLD activity on the Pd plate, the Pd plate was transferred to a 12-well plate. The enzyme reaction was started by adding a GLD activity measuring reagent kept at 25 ° C. in advance to the 12-well plate. Sampling was performed over time, and the absorbance at 600 nm was measured with a plate reader (SpectraMax Plus 384, manufactured by Molecular Devices). The glucose dehydrogenase activity measuring reagent was 1.00 mL of 100 mM potassium phosphate buffer (pH 7.0), 1.00 mL of 1M D-glucose solution, 0.61 mL of MilliQ water, 3 mM 2,6-dichlorophenolindophenol. It is a mixed solution of 0.14 mL and 3 mM 1-methoxy-5-methylphenadium methyl sulfate 0.20 mL. As a control, measurement was performed in the same manner as described above using glucose dehydrogenase not linked with a metal-binding peptide. Each was measured twice and the average value was calculated. The results are shown in FIG. On the vertical axis, the GLD residual activity on the Pd plate is expressed as a relative value with the average value of the control being 1.

  As a result, when compared with average values, GLD-N1C1 'had a GLD residual activity on the Pd plate of about 3.8 times that of GLD not linked with a metal-binding peptide. Therefore, the metal-binding glucose dehydrogenase can increase the metal-binding ability by the linked metal-binding peptide, and can be appropriately bound on the Pd plate, and can exhibit high residual activity on the Pd plate. It seems to have become.

DESCRIPTION OF SYMBOLS 1 ... Protein, 2 ... Metal binding peptide, 3 ... Electrode, 4 ... Metal binding protein

Claims (14)

A binder comprising the following properties (i), (ii), (iii) and (iv) and comprising a metal-binding peptide:
(I) has gold and platinum group metal binding ability;
(Ii) is a basic peptide;
(Iii) is a hydrophilic peptide;
(Iv) It has an orientation control ability.   The binding agent according to claim 1, comprising a metal-binding peptide having a total ratio of R and K in the whole peptide of at least 30%.   The binding agent according to claim 1 or 2, comprising a 6 amino acid metal binding peptide. The binder according to any one of claims 1 to 3, which has the following properties (v), (vi) and (vii):
(V) the isoelectric point is 8.5 to 13.5;
(Vi) the hydrophilicity / hydrophobicity index is -0.200 to -4,000;
(Vii) It consists of 4 to 14 amino acids. A binding agent comprising a peptide having the following amino acid sequence (a) or (b) and having a binding ability to a metal:
(A) the amino acid sequence according to any one of (1) to (17) below:
(1) an amino acid sequence consisting of K, K, R, E, V and R;
(2) an amino acid sequence consisting of V, Y, N, K, R and K;
(3) an amino acid sequence consisting of S, R, A, A, K and Y,
(4) an amino acid sequence consisting of Q, K, R, K, V and V;
(5) an amino acid sequence consisting of K, G, R, G, R and V;
(6) an amino acid sequence consisting of K, R, K, A, A and M;
(7) an amino acid sequence consisting of K, T, R, G, V and K;
(8) an amino acid sequence consisting of K, Q, K, K, T and T;
(9) an amino acid sequence consisting of R, T, R, N, R and S,
(10) an amino acid sequence consisting of T, Q, K, G, R and K;
(11) an amino acid sequence consisting of K, G, A, K, K and V,
(12) an amino acid sequence consisting of K, K, T, S, K and G;
(13) an amino acid sequence consisting of K, T, R, M, R and G;
(14) an amino acid sequence consisting of L, K, D, K, K and K;
(15) an amino acid sequence consisting of R, G, Y, K, K and G;
(16) an amino acid sequence consisting of R, K, G, N, K and A,
(17) An amino acid sequence consisting of R, V, G, R, K and G, wherein one or several amino acids are deleted, substituted or added in the amino acid sequence described in (b) (a). The binding agent according to claim 5, which comprises a peptide having the following amino acid sequence (c), (d) or (e) and has a binding ability to a metal:
(C) the amino acid sequence according to any one of SEQ ID NOs: 1 to 17;
(D) a reverse sequence of the amino acid sequence described in (c);
(E) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in (c) or (d).   The binder according to any one of claims 1 to 6, comprising an amino acid selected from K, R, G, V, T, A, Q, S, Y, N, M, L, D or E.   A metal-binding protein obtained by linking the binding agent according to any one of claims 1 to 7 to a protein.   The metal-binding protein according to claim 8, wherein a binder is linked to the N-terminus and / or C-terminus of the protein.   The metal-binding protein according to claim 8 or 9, wherein the protein is an antibody or an enzyme.   The metal binding protein according to any one of claims 8 to 10, wherein the protein is an oxidoreductase.   A measurement reagent composition comprising the metal binding protein according to any one of claims 8 to 11.   The measuring method of the measuring object in a sample which uses a metal binding protein in any one of Claims 8-11.   The measurement method according to claim 13, wherein the measurement object is glucose.

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