JP6623406B2 - Binder - Google Patents

Description

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

  Electrodes in which proteins such as antibodies or enzymes are immobilized on the surfaces of the electrodes are used for biosensors and biocells. For the immobilization, a method using a cross-linking agent is known, but there is a problem that it is difficult to control the orientation of the protein (Patent Document 1).

  An immobilization method for controlling the orientation is also known, but there is a problem that the electrode constituting base material is limited to a carbon base material, or the enzyme needs to have a specific structure, and the 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

  In immobilizing a protein on an electrode, an immobilization method that can control orientation and is versatile has been demanded. The present invention has been made in view of the above, and provides a binding agent having a metal binding ability and a metal binding protein linked with the binding agent.

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

That is, the present invention relates to the following aspects [1] to [14].
[1] A binder comprising a metal-binding peptide having the following characteristics (i), (ii), (iii) and (iv):
(I) having a gold and platinum group metal binding ability;
(Ii) a basic peptide;
(Iii) a hydrophilic peptide;
(Iv) It has an orientation controllability.
[2] The binding agent according to [1], comprising a metal-binding peptide in which the total ratio of R and K to the entire 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], having the following properties (v), (vi) and (vii):
(V) an isoelectric point of 8.5 to 13.5;
(Vi) the hydrophilic / hydrophobic index is from -0.200 to -4.00;
(Vii) consists of 4 to 14 amino acids.
[5] A binder comprising a peptide having the following amino acid sequence of (a) or (b), and having a binding ability to a metal:
(A) an amino acid sequence according to any of the following (1) to (17);
(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 (a).
[6] The binder according to [5], comprising a peptide having the following amino acid sequence of (c), (d) or (e), and having a binding ability to a metal:
(C) the amino acid sequence of any one of SEQ ID NOs: 1 to 17;
(D) the reverse sequence of the amino acid sequence according to (c);
(E) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence according to (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 and E. Binder.
[8] A metal binding protein in which the binding agent according to any one of [1] to [7] is linked to the protein.
[9] The metal-binding protein according to [8], wherein a binding agent is linked to the N-terminal and / or C-terminal 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, using the metal-binding protein according to any one of [8] to [11].
[14] The measurement method according to [13], wherein the measurement object is glucose.

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

It is a schematic diagram of a metal binding protein immobilized electrode. From the left, the N-terminus and C-terminus of the protein, and the immobilization of the metal-binding protein in which a metal-binding peptide is linked to the N-terminus and the C-terminus are shown. It is a schematic diagram of metal binding esterase Est-N1-17 and Est-C1-17. It is an evaluation result of the binding ability of metal binding esterase. It is an evaluation result of the orientation control ability 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 binding agent of the present invention comprises a metal binding peptide having the following properties.
(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 controllability.

The binding agent of the invention preferably consists of a metal binding peptide having the following properties:
(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 (Theoretic pI) calculated by ProtParam tool (http://web.expasy.org/protparam/) of ExPASy.
(Vi) The hydrophilicity / hydrophobicity index is -0.200 to -4.0000, preferably -0.500 to -3.500, and more preferably -0.800 to -3.200.
The hydrophilic / hydrophobic index is a grand average of hydropathicity (GRAVY) calculated by ProtParam Tool (http://web.expasy.org/protparam/) of ExPASy.
(Vii) a binder consisting of 4 to 14 amino acids, preferably 5 to 13 amino acids or 6 to 12 amino acids, and more preferably at most 10 amino acids, 9 amino acids, 8 amino acids or 7 amino acids. More preferably, it consists of 6 amino acids.

  The binding agent 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 of (a) or (b), and has 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;
In the amino acid sequences described in (1) to (17), the order of the constituent amino acids is not limited. More preferably, the sequence is the sequence described above, or the reverse sequence. More preferably, it consists of a peptide having the amino acid sequence of any one of (1) to (17), and has a metal binding ability. It preferably has at least one of the characteristics (i) to (viii). The amino acids described in (1) to (17) are all represented by one letter.
(B) a peptide having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence described in the above (a), and having a metal binding ability. The number is preferably three, and more preferably two.

The binding agent of the present invention preferably comprises a peptide having the following amino acid sequence (c), (d) or (e), and has a binding ability to a metal. It preferably has at least one of the characteristics (i) to (viii).
(C) the amino acid sequence of any one of SEQ ID NOs: 1 to 17;
(D) The 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 according to (c) or (d). The number is preferably three, and more preferably two.

The binding agent of the present invention is preferably composed of a peptide having the following amino acid sequence (f), and has binding ability to metal.
(F) An amino acid sequence in which two of the amino acid sequences according to (a), (b), (c), (d) or (e) are tandem-bonded. The combination of the two tandem-binding sequences 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 sequence.

  The binding agent of the present invention preferably comprises an amino acid selected from K, R, G, V, T, A, Q, S, Y, N, M, L, D or E. It preferably does not contain at least one amino acid selected from C, H, P, W, I and 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 genetic recombination or a peptide obtained by synthesis. Genetic recombination may be performed by a known method using the nucleotide sequence encoding the amino acid sequence described in the above (a), (b), (c), (d), (e) or (f). .

  The binding agent of the present invention may be any peptide that has 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, the platinum group metal can be exemplified by platinum, palladium, rhodium, ruthenium or iridium, with palladium being more preferred. The binder of the present invention can bind to an electrode using the metal as a conductive substance, and can immobilize an enzyme, an antibody, or the like used for measurement on the electrode.

  By linking the binding agent to a protein, a metal binding protein can be produced. The binding agent is preferably linked to the N-terminal and / or C-terminal of the protein. When a binding agent is linked to the N-terminus of the protein, it may be linked to the N-terminus of a mature protein that does not contain a secretory signal sequence. Further, there may be a linker peptide between the protein and the binder. The ligation method may be a total synthesis of the entire metal binding protein or a genetic engineering technique. The binding agent of the present invention hardly adversely affects the structure of the protein even when linked to the protein, and can maintain the properties of the protein.

  For example, a genetic engineering technique prepares a vector into which a base sequence encoding a metal binding protein has been inserted. 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 contains the base sequence as an insert. As the transformant, for example, prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as fungi (yeast, Ascomycetes such as Aspergillus, basidiomycetes, etc.), insect cells, mammalian cells, and the like are used. Can be. The vector may be maintained in a transformed cell in a state like a plasmid, or may be maintained by being integrated into a chromosome. Furthermore, a host can be appropriately selected as necessary, such as necessity of sugar chains, unnecessary one, secretory production, and the like. When a host is to secrete and produce a metal-binding protein in which a binding agent is linked to the N-terminal side of the protein, the base sequence is designed so that a secretory signal sequence exists before the sequence of the binding agent. Alternatively, a secretory signal sequence on the vector side may be used. When a host produces a metal-binding protein in which a binding agent is linked to the C-terminal side of the protein, a gene sequence encoding the binding agent is inserted before the termination 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 binding agent may be a glycoprotein. The protein is preferably an enzyme or an antibody. When the binding agent is linked to the protein, a metal-binding enzyme or a metal-binding antibody is obtained. The protein can be exemplified by a hydrolase, a transferase or an oxidoreductase. When the above-mentioned binder is linked thereto, a metal-binding hydrolase, a metal-binding transferase or a metal-binding 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 oxidase and dehydrogenase. For example, an enzyme that catalyzes an oxidation reaction using glucose, lactic acid, cholesterol, or alcohol as a substrate can be exemplified. 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 of SEQ ID NOs: 18 to 22 can be used. A metal-binding protein is produced by linking the sequence of the binding agent according to any one of (a) to (f) to the N-terminal and / or C-terminal of the amino acid sequence of SEQ ID NOS: 18 to 22. Is also good. An amino acid sequence having at least 80%, 85%, 90%, 95% or 98% similarity to any of the amino acid sequences described in SEQ ID NOs: 18 to 22 may be used. An amino acid sequence in which one or more amino acids are deleted, substituted or added in any of the amino acid sequences described in SEQ ID NOs: 18 to 21 may be used. The number of mutations is preferably at most 60, 55, 50, 40, 30, 20, 15, 10, 5, 3, or 2 at most. When the nucleotide sequences encoding those amino acid sequences are used, the metal-binding protein of the present invention can be produced by the genetic engineering technique.

  By immobilizing the metal-binding protein of the present invention on the electrode by chemical bonding, a metal-binding protein-immobilized electrode can be produced. The metal-binding protein of the present invention can be easily bound to an electrode provided with the above-mentioned metal conductive substance via a binder portion, and can be immobilized with orientation. FIG. 1 shows a schematic diagram of the metal-binding protein-immobilized electrode.

  A biosensor including an electrode having the metal-binding protein of the present invention immobilized thereon and an insulating substrate can be manufactured. For example, an electrochemical biosensor can be manufactured by including a step of forming an electrode system on an insulating substrate using a method such as screen printing or vapor deposition, 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 of a type that detects a current value, an ion, a coloring intensity, a frequency change, and the like. In addition, it may contain mediators, buffers or stabilizers.

  Examples of the mediator include a proteinaceous electron mediator such as heme, a ferricyanide compound, a quinone compound, an osmium compound, a phenazine compound, and a phenothiazine compound. The buffer may be any buffer that can be adjusted to the target reaction environment. Examples of the stabilizer include bovine serum albumin (BSA) or ovalbumin, sugars or sugar alcohols having no effect on the molecular identification element, carboxyl group-containing compounds, alkaline earth metal compounds, ammonium salts, sulfates or proteins. Can be illustrated.

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

[Example 1]
(Acquisition of metal-binding peptide)
In order to obtain a metal-binding peptide having a metal-binding ability, a random peptide library consisting of a 6-amino acid sequence was constructed by phage display and biopanning was performed. Specifically, the cycles (1) to (3) or (4) were performed under the following washing conditions I, II or III. Finally, 17 types of phage that strongly bind to the palladium (Pd) plate were obtained, and DNA sequence analysis was performed. Table 1 shows the amino acid sequences of 17 types of metal binding peptides (P1 to P17) determined from the analyzed DNA sequence. For each amino acid sequence, the theoretical isoelectric point (Theoretic pI) and the hydrophilic / hydrophobic index (Grand average of hydropathicity (GRAVY)) were determined using the ExPASy ProtParam tool (http://web.expasy.org/protparam/). Calculated and described in Table 1. All were basic peptides. Table 2 summarizes the Au, Pt or Pd binding peptides described in Non-Patent Documents 1 and 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
Washing 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
Washing 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: A T7 phage suspension displaying a random peptide is prepared.
Step 2: The Pd plate is immersed in the T7 phage suspension at 37 ° C. for 1 hour to allow the Td phage to bind to the Pd plate.
Step 3 (washing step): The Pd plate to which the T7 phage is bound is immersed in a buffer solution of each washing condition at 37 ° C. for 5 minutes to wash. The washing is performed for each washing condition.
Step 4: The washed Pd plate is immersed in a culture solution of Escherichia coli BL21 strain, and T7 phage remaining on the Pd plate is infected with Escherichia coli BL21 and amplified.
Step 5: Amplify 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 of SEQ ID NOs: 1 to 17 were linked to the N-terminal or C-terminal of the esterase (Est) of SEQ ID NO: 18, respectively. Various kinds of metal binding Ests were produced. 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 the gene expressing Est described in SEQ ID NO: 18 was inserted was used.

  In order for Est-N1 to be expressed, a base sequence encoding the amino acid sequence of SEQ ID NO: 1 was inserted before the start codon of the Est gene in the vector, and an expression vector pEst-N1 containing the full length of the Est-N1 gene was obtained. Produced. Similarly for Est-N2 to 17, expression vectors pEst-N2 to 17 containing the full length of Est-N2 to 17 genes were similarly prepared.

  In order to express Est-C1, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 is inserted before the stop codon of the Est gene in the vector, and an expression vector pEst-C1 containing the full length of the Est-C1 gene is obtained. Produced. Similarly for Est-C2-17, expression vectors pEst-C2-17 containing the full length of Est-C2-17 gene were prepared.

  Escherichia coli was transformed with each of the above vectors, and a total of 34 recombinant Escherichia coli producing Est-N1 to 17 or Est-C1 to 17 were produced. Each recombinant strain was cultured in a liquid medium containing ampicillin and collected. Each of the collected recombinant Escherichia coli was sonicated to recover each cell-free extract (CFE) containing Est-N1 to 17 or Est-C1 to 17. After heat treatment of each CFE, purification was performed using a His-tag fusion protein purification column, and a total of 34 samples of purified Est-N1 to 17 or purified Est-C1 to 17 were collected. When each purified enzyme was confirmed by SDS-PAGE, contaminating proteins could be removed. Furthermore, all metal-binding Ests had the same activity as Est not linking the metal-binding peptide. Even when the metal-binding peptide of the present invention was linked to the N-terminus and / or C-terminus of Est, inactivation by the ligation was not observed, indicating that the enzyme activity could be maintained. FIG. 2 shows a schematic diagram of each metal binding Est.

(2) Evaluation of Binding Ability of Metal-Binding Esterase For each of the 34 types of metal-binding Est obtained in (1) of Example 2, the binding ability to palladium was evaluated. A 1 cm × 1 cm plastic plate (Pd plate) with one side palladium-plated was fixed to a 24-well plate with a double-sided tape with the plated side up. 800 μL of a 10 μg / ml Est-N1 solution was added to the plate, and the plate was 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, which was kept at 37 ° C. in advance. 50 μL of 100 mM p-nitrophenylacetic acid was added to the tube to start the enzyme reaction. Every 5 minutes, 100 μL was sampled into a 96-well plate, and the absorbance at 405 nm was measured with a plate reader. Est-N2 to 17 and Est-C to 17 were measured in the same manner as described above. As a control, the measurement was carried out in the same manner as above using Est to which the metal-binding peptide was not linked. The results are shown in FIG. On the horizontal axis, the 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 esterase residual activity on the Pd plate is represented by a relative value with the control set to 1.

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

[Example 3]
(Metal-binding o-acetylserine sulfhydrylase)
(1) Preparation of metal-binding o-acetylserine sulfhydrylase Metal-binding peptide P1, 2, 3, 4, 9, or 13 having the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 9, or 13 Was linked to the N-terminus or the C-terminus of o-acetylserine sulfhydrylase (OASS) described in SEQ ID NO: 19 to prepare 12 types of metal-binding OASS. Each metal-binding 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 were produced by genetic engineering techniques. As the vector, a His-tag fusion expression vector into which the OASS-expressing gene described in SEQ ID NO: 19 was inserted was used.

  In order for OASS-N1 to be expressed, a base sequence encoding the amino acid sequence of SEQ ID NO: 1 was inserted before the start codon of the OASS gene in the vector, and an expression vector pOASS-N1 containing the entire OASS-N1 gene was obtained. Produced. Similarly, for OASS-N2, OASS-N3, OASS-N4, OASS-N9 or OASS-N13, expression vectors pOASS-N3, pOASS-N4, pOASS-N9 or pOASS-N13 were respectively produced.

  In order for OASS-C1 to be expressed, a base sequence encoding the amino acid sequence of SEQ ID NO: 1 is inserted before the stop codon of the OASS gene in the vector, and the expression vector pOASS-C1 containing the entire OASS-C1 gene is obtained. Produced. Similarly, expression vectors pOASS-C2, pOASS-C3, pOASS-C4, pOASS-C9 or pOASS-C13 were similarly prepared for OASS-C2, OASS-C3, OASS-C4, OASS-C9 or OASS-C13.

  Escherichia coli was transformed with each of the above vectors, and OASS-N1, OASS-N2, OASS-N3, OASS-N4, OASS-N9, OASS-N13, OASS-C1, OASS-C2, OASS-C3, OASS-C4. , OASS-C9 or OASS-C13-producing recombinant Escherichia coli totaling 12 species. Each recombinant strain was cultured in a liquid medium containing ampicillin and collected. Each of the collected recombinant Escherichia coli was sonicated and crushed by 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, it refine | purified with the His tag fusion protein purification column, and refine | 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. When each purified enzyme was confirmed by SDS-PAGE, contaminating proteins could be removed.

  In addition, a metal-binding OASS in which a palladium-binding peptide: SPHPGPY described in Non-Patent Document 1 was linked to the N-terminal or C-terminal of OASS was also prepared in the same manner as above and used as a control.

(2) Evaluation of the ability to control the orientation of metal-binding o-acetylserine sulfhydrylase For each of the 12 metal-binding OASSs obtained in (1) of Example 3, a NAPiCOS system (manufactured by Nihon Dempa Kogyo Co., Ltd.) Was used to evaluate the orientation controllability. First, with respect to a QCM sensor (manufactured by Nippon Dempa Kogyo Co., Ltd.), a gold electrode surface of a quartz oscillator was subjected to palladium vapor deposition to produce a palladium film-formed QCM sensor. A 100 μg / mL OASS-N1 solution was added to palladium of the sensor, and allowed to stand at room temperature for 1 hour, so that OASS-N1 was bound to the palladium surface. Next, the sensor was washed with MilliQ water to remove unbound OASS-N1, thereby producing an OASS-N1 immobilized sensor chip. 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. The same operation is performed 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. Was performed and the change in frequency was measured. The results are shown in FIG. In FIG. 4, the metal-binding peptides P1, P2, P3, P4, P9 or P13 linked to the N-terminus or C-terminus are represented by P1, P2, P3, P4, P9 or P13, and the control is represented by PC. expressed. The dotted line represents the frequency change of the metal-binding OASS having a metal-binding peptide linked to the N-terminal, and the solid line represents the frequency of the metal-binding OASS having a metal-binding peptide linked to the C-terminal. It represents a change.

  When a metal-binding peptide is linked to the N-terminal side of a protein, it binds to the metal at the N-terminal side, whereas when a metal-binding peptide is linked to the C-terminal side, it binds to the metal at the C-terminal side. OASS has an active site near the C-terminus, so theoretically, if it binds to the metal at the C-terminus, SAT does not easily act, so the measured value is small. If it binds to the metal at the N-terminus, SAT acts on the active site. It is considered that the measured value becomes large because the measurement is easy. Therefore, comparing the measured values of OASS-N1 and OASS-C1, if the frequency change of OASS-N1 is larger, it can be determined that the orientation is controlled 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- In N9 and OASS-C9 (P9) or OASS-N13 and OASS-C13 (P13), the frequency change was greater when the metal-binding peptide was linked to the N-terminal. On the other hand, in the control (PC), no difference was observed between those in which the metal-binding peptide was linked to the N-terminal side and the C-terminal side. That is, it was clarified that the orientation was not substantially 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 kinds of metal-binding glucose dehydrogenases in which a binding peptide having the amino acid sequence of SEQ ID NO: 1 or its reverse sequence was linked were prepared. The first is GLD-N1 in which a binding peptide having the amino acid sequence of 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 of SEQ ID NO: 1 is linked to the N-terminus of GLD and a binding peptide having the reverse sequence of the amino acid sequence of SEQ ID NO: 1 is linked to the C-terminus. is there. GLD-N1, GLD-C1 'and GLD-N1C1' were all produced by genetic engineering techniques. As the vector, an expression vector into which the gene for 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 WO2006 / 101239 is replaced by a gene encoding MLFSLAFLSALSLATASPAGRA.

  A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 was inserted after the signal gene sequence in the vector so that GLD-N1 was expressed, thereby preparing an expression vector pGLD-N1 containing the entire GLD-N1 gene. .

  In order to express GLD-C1 ′, a nucleotide 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 includes the entire length of the GLD-C1 ′ gene. 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 before the stop codon of the GLD gene in the pGLD-N1 to obtain the full-length GLD-N1C1 ′ gene. The expression vector pGLD-N1C1 ′ containing was prepared.

  According to the method described in Example 4 of WO 2006/101239 pamphlet, each vector was used to transform Aspergillus oryzae NS4 strain, and GLD-N1, GLD-C1 'or GLD-N1C1'-producing recombinant Aspergillus oryzae 3 was transformed. Seeds were made. Each recombinant strain was cultured in a liquid medium, and the culture supernatant was collected. Each culture supernatant was purified by an anion exchange column and confirmed by SDS-PAGE. As a result, a single band of GLD-N1, GLD-C1 'or GLD-N1C1' could be confirmed. Furthermore, all the metal-binding GLDs had the same activity as the GLD not linked to the metal-binding peptide. Even when the metal-binding peptide of the present invention was linked to the N-terminus and / or C-terminus of GLD, inactivation by the ligation was not observed, indicating that the enzyme activity could be maintained. FIG. 5 shows a schematic diagram of each metal-binding GLD.

(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), one side of which was palladium-plated, was fixed to a 12-hole plate with a double-sided tape with the plated side up. 1 mL of a 7 μg / ml GLD-N1C1 ′ solution was added to the plate, and the plate was allowed to stand at 37 ° C. for 1 hour, so that GLD-N1C1 ′ was bound to the 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 '.

  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 the reagent for measuring GLD activity, which was kept at 25 ° C. in advance, to the 12-well plate. Samples were taken over time, and the absorbance at 600 nm was measured using a plate reader (SpectraMax Plus384, manufactured by Molecular Devices). The reagent for measuring glucose dehydrogenase activity was 1.00 mL of 100 mM potassium phosphate buffer (pH 7.0), 1.00 mL of 1 M D-glucose solution, 0.61 mL of MilliQ water, and 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 to a metal-binding peptide. Each was measured twice and the average value was calculated. The results are shown in FIG. The vertical axis represents the relative activity of the GLD residual activity on the Pd plate, with the average value of the control as 1.

  As a result, comparing GLD-N1C1 'with an average value, GLD residual activity on the Pd plate was about 3.8 times that of GLD not linked to a metal-binding peptide. Therefore, the metal-binding glucose dehydrogenase can enhance the metal-binding ability and properly bind to the Pd plate by the linked metal-binding peptide, and can exhibit high residual activity on the Pd plate. It seems that it has become.

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

Claims (8)

A metal-binding peptide having the following characteristics (i), (ii), (iii) and (iv), and having the amino acid sequence of any one of SEQ ID NOs: 1 to 17:
(I) having an ability to bind to palladium;
(Ii) the total proportion of R and K in the total peptide is at least 30%;
(Iii) the isoelectric point is between 9.5 and 12.5; and
(Iv) the hydrophilic / hydrophobic index is -0.800 to -3.200 ;
A binder consisting of   A metal-binding protein comprising the protein and the binding agent according to claim 1 linked thereto.   The metal-binding protein according to claim 2, wherein a binding agent is linked to the N-terminal and / or C-terminal of the protein.   4. The metal binding protein according to claim 2, wherein the protein is an antibody or an enzyme.   The metal-binding protein according to any one of claims 2 to 4, wherein the protein is an oxidoreductase.   A measurement reagent composition comprising the metal-binding protein according to any one of claims 2 to 5.   A method for measuring an object to be measured in a sample, using the metal-binding protein according to any one of claims 2 to 5. The measurement method according to claim 7, wherein the measurement object is glucose.

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