WO2017026505A1 - Agent de liaison - Google Patents

Agent de liaison Download PDF

Info

Publication number
WO2017026505A1
WO2017026505A1 PCT/JP2016/073537 JP2016073537W WO2017026505A1 WO 2017026505 A1 WO2017026505 A1 WO 2017026505A1 JP 2016073537 W JP2016073537 W JP 2016073537W WO 2017026505 A1 WO2017026505 A1 WO 2017026505A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
binding
metal
identification element
Prior art date
Application number
PCT/JP2016/073537
Other languages
English (en)
Japanese (ja)
Inventor
高史 宅見
洋行 今中
今村　維克
晴香 的場
Original Assignee
池田食研株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2015158726A external-priority patent/JP6623406B2/ja
Priority claimed from JP2015158727A external-priority patent/JP2017037015A/ja
Application filed by 池田食研株式会社 filed Critical 池田食研株式会社
Publication of WO2017026505A1 publication Critical patent/WO2017026505A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

Definitions

  • the present invention relates to a binder having a metal binding ability, a molecular identification element fixed electrode, and the like.
  • Electrodes in which molecular identification elements such as antibodies or enzymes are immobilized on the surface of electrodes are used for biosensors and biocells.
  • a method using a crosslinking agent is known for immobilization, there is a problem that it is difficult to control the orientation of the enzyme which is a molecular identification element (Patent Document 1).
  • Non-patent Documents 1 and 2 disclose several metal-binding peptides.
  • the present invention has been made in view of the above, a binding agent having a metal binding ability, a metal binding molecular identification element linked with the binding agent, a molecular identification element fixed electrode on which the molecular identification element is fixed, A method for producing the electrode, a biosensor including the electrode, and a biobattery including the electrode are provided.
  • the inventors have found a peptide having a metal-binding ability (metal-binding peptide), that the metal-binding peptide can be used as a binding agent between a protein and a metal, and that the peptide is linked to a molecular identification element composed of a protein. And found that it becomes a metal-binding molecular identification element. Furthermore, it has been found that by using the metal binding molecular identification element, the molecular identification element can be fixed to the electrode with orientation. Furthermore, the manufacturing method of the molecule
  • 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 (v) 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.
  • 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 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, K,
  • [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 set forth in 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.
  • a metal-binding molecular identification element wherein the binding agent according to any one of [1] to [7] is linked to a molecular identification element composed of a protein.
  • the manufacturing method of the molecular identification element fixed electrode including the process fixed by coupling
  • a biosensor for measuring a measurement object in a sample solution comprising an insulating substrate and the electrode according to any one of [17] to [21].
  • the biosensor according to [22] which is for glucose measurement.
  • a biocell comprising the electrode according to any one of [17] to [21].
  • a binder comprising a peptide having a metal binding ability
  • a metal-binding molecular identification element can be obtained by linking the binder to a molecular identification element composed of a protein such as an antibody or an enzyme. Since the metal-binding molecular identification element easily binds to a metal, it can be easily fixed to an electrode, and the molecular identification element can be fixed with an orientation in a desired direction. Furthermore, according to the present invention, the protein shape of the molecular identification element is stabilized on the electrode, and inactivation due to structural change can be suppressed. Therefore, the amount of protein on the electrode can be suppressed, and the cost can be reduced.
  • FIG. 2 is a schematic diagram of metal-binding esterases Est-N1 to 17 and Est-C1 to 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.
  • FIG. 2 is a schematic diagram of metal-binding glucose dehydrogenases 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 (I) of the present invention comprises a metal-binding peptide having the following properties (i) to (iv).
  • the binding agent (I) of the present invention is preferably a metal-binding peptide (v) in which the total ratio of arginine (R) and lysine (K) in the whole 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 binder (I) of the present invention more preferably comprises a metal-binding peptide having the following properties (vi) to (viii).
  • 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/).
  • the hydrophilicity / hydrophobicity index is ⁇ 0.200 to ⁇ 4.0000, preferably ⁇ 0.500 to ⁇ 3.500, more preferably ⁇ 0.800 to ⁇ 3.200.
  • hydrophilicity / hydrophobicity index is a hydrophilicity / hydrophobicity index (GRAVY) calculated by ExPASy's ProtParam tool (http://web.expasy.org/protparam/).
  • GRAVY hydrophilicity / hydrophobicity index
  • a binding agent comprising 4 to 14 amino acids, preferably 5 to 13 amino acids or 6 to 12 amino acids, and more preferably 10 amino acids, 9 amino acids, 8 amino acids or 7 amino acids. More preferably, it consists of 6 amino acids.
  • the binding agent (II) of the present invention is also composed of 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.
  • An amino acid sequence consisting of K, T, R, G, V and K An amino acid sequence consisting of K, Q, K, K, T, and T.
  • An amino acid sequence consisting of R, T, R, N, R and S. An amino acid sequence consisting of T, Q, K, G, R and K.
  • An amino acid sequence consisting of K, G, A, K, K and V. An amino acid sequence consisting of K, K, T, S, K and G.
  • An amino acid sequence consisting of L, K, D, K, K and K. An amino acid sequence consisting of R, G, Y, K, K and G.
  • amino acid sequence consisting of R, K, G, N, K and A 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 (sequence in which the N-terminal and C-terminal are interchanged in the original amino acid 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).
  • the amino acids described in (1) to (17) are all represented by one letter.
  • 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 binder (II) 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 metal. More 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 binder (II) of the present invention is preferably composed of a peptide having the following amino acid sequence (f) and has a binding ability to metal.
  • 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 binders (I) and (II) of the present invention are preferably composed of amino acids selected from K, R, G, V, T, A, Q, S, Y, N, M, L, D or E. More preferably, it does not contain at least one amino acid selected from C, H, P, W, I or F.
  • the method for preparing the peptide constituting the binding agent of the present invention is not particularly limited, and it may be a peptide obtained by gene recombination or a peptide obtained by synthesis.
  • gene recombination is performed by a well-known method using a base sequence encoding the amino acid sequence described in (a), (b), (c), (d), (e) or (f). Just do it.
  • the binding agent of the present invention may be any peptide (metal-binding peptide) having the ability to bind to metal, and any peptide that can chemically bind to metal.
  • the metal to which the binder can bind is preferably gold or a platinum group metal.
  • 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.
  • a metal-binding molecular identification element can be produced by linking the binding agent to a molecular identification element made of protein.
  • the binding agent is preferably linked to the N-terminus and / or C-terminus of the protein.
  • linker peptide between the protein and the binder.
  • a person skilled in the art can appropriately design and prepare such a linker peptide and is usually composed of about 2 to 50 amino acids.
  • 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.
  • a vector into which a base sequence encoding a metal binding protein is inserted is prepared.
  • a host microorganism is transformed using the expression vector to obtain a transformant.
  • 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.
  • 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.
  • the host can be appropriately selected according to the necessity, such as the necessity of the sugar chain, unnecessary, secretory production, etc.
  • the base sequence is designed so that a secretory signal sequence exists before the binding agent sequence.
  • a secretory signal sequence on the vector side may be used.
  • 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.
  • 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 which is a molecular identification element.
  • a metal binding enzyme or a metal binding antibody is obtained.
  • the protein include hydrolase, transferase, and oxidoreductase.
  • metal-bonded hydrolase, metal-bound transferase, or metal-bound oxidoreductase is obtained.
  • the hydrolase can be exemplified by esterase
  • 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.
  • a protein having a known sequence may be used.
  • a protein having the amino acid sequence shown in SEQ ID NOs: 18 to 22 can be used.
  • a metal-binding molecule discriminating element obtained by linking, for example, 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. May be manufactured.
  • 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.
  • the “similarity” in the amino acid sequence is based on the value of Similarity calculated by homology analysis between the amino acid sequences of GENETYX (Genetics). 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 molecular identification element of the present invention can be produced by the genetic engineering technique.
  • the molecular identification element-immobilized electrode can be produced by immobilizing the molecular identification element on the electrode by mounting, adhering or bonding the metal-binding molecular identification element of the present invention to the electrode by chemical bonding.
  • the molecular identification element of the present invention can be easily bonded to the electrode provided with the metal conductive material through the binder portion of the present invention, and the molecular identification element can be fixed with orientation in a desired direction.
  • a schematic diagram of the molecular identification element-immobilized electrode is shown in FIG.
  • a biosensor including an electrode on which the molecular identification element of the present invention is immobilized can be produced.
  • an electrochemical biosensor is manufactured by including a substrate, a counter electrode, a working electrode, and a molecular identification element.
  • the electrode preferably includes a counter electrode and a working electrode, and may include a reference electrode.
  • mediators, buffers or stabilizers may be included.
  • the mediator examples include proteinaceous electron mediators such as heme, ferricyanide compounds, quinone compounds, osmium compounds, phenazine compounds, phenothiazine compounds, and the like.
  • the buffer may be any buffer that can be adjusted to the target reaction environment.
  • 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.
  • the biosensor of the present invention 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 solution. 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.
  • 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.
  • eukaryotic cell-derived glucose dehydrogenase using flavin adenine dinucleotide as a coenzyme is preferable.
  • the bio battery of the present invention may be a battery including the electrode of the present invention.
  • the biobattery according to the present invention includes an anode electrode that performs an oxidation reaction and a cathode electrode that performs a reduction reaction, and includes an electrolyte layer that separates the anode and the cathode as necessary.
  • the above mediator and enzyme electrode are used as the anode electrode, and electrons generated by oxidizing the substrate are taken out to the electrode and protons are generated.
  • an enzyme generally used for the cathode electrode may be used on the cathode side, for example, by using laccase, ascorbate oxidase or bilirubin oxidase, and reacting protons generated on the anode side with oxygen. Generate water.
  • Example 1 (Acquisition of metal-binding peptide)
  • 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.
  • 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 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 property Est is, respectively, Est-N1, Est-N2, Est-N3, Est-N4, Est-N5, Est-N6, Est-N7, Est-N8, Est-N9, Est-N10, Est.
  • 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.
  • Est-N1 a base sequence encoding the amino acid sequence shown 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 full length of the Est-N2-17 gene were prepared.
  • Est-C1 In order to express Est-C1, a base sequence encoding the amino acid sequence described in 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 inserted. Produced. Similarly, for Est-C2-17, expression vectors pEst-C2-17 containing the full 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 to collect 34 samples of purified Est-N1-17 or purified Est-C1-17. When each purified enzyme was confirmed by SDS-PAGE, contaminating proteins could be removed.
  • 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.
  • the Pd plate was transferred to a 5 mL tube containing 4.95 mL PBS which was kept at 37 ° C. in advance. 50 ⁇ L of 100 mM p-nitrophenylacetic acid was added to the tube to start the enzyme reaction. 100 ⁇ L 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 to 17 and Est-C to 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.
  • 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.
  • the residual activity of esterase on the Pd plate is expressed as a relative value with the control being 1.
  • Est-N1-17 and Est-C1-17 had an Est residual activity on the Pd plate of about 1.5 to 4.0 times that of Est not linked with a 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) Preparation 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 Twelve types of metal-binding OASS were prepared by linking these to the N-terminus or C-terminus of o-acetylserine sulfhydrylase (OASS) described in SEQ ID NO: 19.
  • OASS o-acetylserine sulfhydrylase
  • Each metal-bonded OASS is respectively 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.
  • 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.
  • OASS-N1 In order for OASS-N1 to be expressed, a base sequence encoding the amino acid sequence described in SEQ ID NO: 1 is inserted before the start codon of the OASS gene in the vector, and an expression vector pOASS-N1 containing the full length of the OASS-N1 gene is obtained. Produced. Similarly, for OASS-N2, OASS-N3, OASS-N4, OASS-N9 and OASS-N13, expression vectors pOASS-N3, pOASS-N4, pOASS-N9 and pOASS-N13 were prepared.
  • OASS-C1 In order to express OASS-C1, a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is inserted before the stop codon of the OASS gene in the vector, and an expression vector pOASS-C1 containing the full length of the OASS-C1 gene is obtained. 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 is sonicated, and OASS-N1, OASS-N2, OASS-N3, OASS-N4, OASS-N9, OASS-N13, OASS-C1, OASS-C2, OASS-C3, OASS
  • CFE cell-free extract
  • Non-Patent Document 1 a metal-binding OASS in which SPHPGPY was linked to the N-terminal or C-terminal of OASS was also prepared in the same manner as described above and used as a control.
  • OASS-N1 On the palladium of the sensor, 100 ⁇ g / 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 ⁇ g / mL serine acetyltransferase (SAT) solution was flowed, and then a 100 ⁇ g / mL SAT solution was flowed to interact with OASS-N1 on the sensor, and each frequency change was measured.
  • SAT serine acetyltransferase
  • 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
  • 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.
  • a metal-binding peptide 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.
  • 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.
  • 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 the orientation control is performed by the metal-binding peptide.
  • 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
  • OASS-N13 and OASS-C13 P13
  • 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.
  • 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).
  • GLD-C1 ′ a binding peptide having the reverse sequence of the amino acid sequence described in SEQ ID NO: 1 is linked to the C-terminal
  • 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-terminus 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-terminus.
  • GLD-N1, GLD-C1 ′ and GLD-N1C1 ′ were all produced by a genetic engineering technique.
  • 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.
  • GLD-N1 In order to express GLD-N1, a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 was inserted after the signal gene sequence in the vector to prepare an expression vector pGLD-N1 containing the full length of the GLD-N1 gene. .
  • GLD-C1 ′ 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 the GLD-C1 ′ gene is included. An expression vector pGLD-C1 ′ was prepared.
  • GLD-N1C1 ′ In order to express GLD-N1C1 ′, a base sequence encoding the reverse sequence of the amino acid sequence shown 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 was prepared.
  • 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.
  • 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.
  • 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 (SpectraMaxMPlus384, 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.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Emergency Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Food Science & Technology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Dans le cadre de l'immobilisation d'éléments de reconnaissance moléculaire sur des électrodes, il existe une demande pour un procédé d'immobilisation apte à réguler l'orientation ainsi que pour un procédé d'immobilisation polyvalent. La présente invention concerne, conformément à la description faite ci-dessus, un agent de liaison apte à se lier au métal, un élément de reconnaissance moléculaire se liant au métal couplé à l'agent de liaison, une électrode d'immobilisation d'élément de reconnaissance moléculaire sur laquelle l'élément de reconnaissance moléculaire est immobilisé, un procédé de fabrication de l'électrode, un capteur incluant l'électrode et un procédé de fabrication du capteur. Les inventeurs de la présente invention ont découvert un peptide apte à se lier au métal (un peptide se liant au métal) et ont découvert que le peptide se liant au métal peut être utilisé comme agent de liaison entre une protéine et un métal et qu'un élément de reconnaissance moléculaire se liant au métal peut être fabriqué en couplant le peptide à un élément de reconnaissance moléculaire comprenant une protéine. Il a en outre été découvert qu'en utilisant l'élément de reconnaissance moléculaire se liant au métal, l'élément de reconnaissance moléculaire peut être immobilisé sur une électrode de façon orientée. Un procédé de fabrication d'une électrode d'immobilisation d'élément de reconnaissance moléculaire faisant intervenir le procédé d'immobilisation a également été découvert, permettant ainsi de mettre en place la présente invention.
PCT/JP2016/073537 2015-08-11 2016-08-10 Agent de liaison WO2017026505A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2015-158727 2015-08-11
JP2015158726A JP6623406B2 (ja) 2015-08-11 2015-08-11 結合剤
JP2015158727A JP2017037015A (ja) 2015-08-11 2015-08-11 分子識別素子固定化電極
JP2015-158726 2015-08-11

Publications (1)

Publication Number Publication Date
WO2017026505A1 true WO2017026505A1 (fr) 2017-02-16

Family

ID=57984218

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/073537 WO2017026505A1 (fr) 2015-08-11 2016-08-10 Agent de liaison

Country Status (1)

Country Link
WO (1) WO2017026505A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025121A2 (fr) * 2001-09-18 2003-03-27 Bioexpertise, Llc Peptide ou petite molecule derive de proteines de liaison de l'igf
JP2004215514A (ja) * 2003-01-09 2004-08-05 Canon Inc 有機物固定化基体
US20060246426A1 (en) * 2003-09-26 2006-11-02 Biohesion, Inc. Recombinant fusion proteins with high affinity binding to gold and applications thereof
JP2007197435A (ja) * 2005-12-28 2007-08-09 Canon Inc 金結合性タンパク質
JP2008076388A (ja) * 2006-08-23 2008-04-03 Canon Inc 酵素電極、酵素電極の製造方法、およびこれを用いたセンサ、燃料電池
JP2010107491A (ja) * 2008-09-30 2010-05-13 Olympus Corp バイオセンサ
JP2014205626A (ja) * 2013-04-11 2014-10-30 学校法人東京薬科大学 白金結合剤、及びその利用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025121A2 (fr) * 2001-09-18 2003-03-27 Bioexpertise, Llc Peptide ou petite molecule derive de proteines de liaison de l'igf
JP2004215514A (ja) * 2003-01-09 2004-08-05 Canon Inc 有機物固定化基体
US20060246426A1 (en) * 2003-09-26 2006-11-02 Biohesion, Inc. Recombinant fusion proteins with high affinity binding to gold and applications thereof
JP2007197435A (ja) * 2005-12-28 2007-08-09 Canon Inc 金結合性タンパク質
JP2008076388A (ja) * 2006-08-23 2008-04-03 Canon Inc 酵素電極、酵素電極の製造方法、およびこれを用いたセンサ、燃料電池
JP2010107491A (ja) * 2008-09-30 2010-05-13 Olympus Corp バイオセンサ
JP2014205626A (ja) * 2013-04-11 2014-10-30 学校法人東京薬科大学 白金結合剤、及びその利用

Similar Documents

Publication Publication Date Title
JP5828554B2 (ja) 蛋白質性電子メディエータ
JP6759418B2 (ja) グルコース測定用バイオセンサ
JP5398004B2 (ja) 改変型グルコース脱水素酵素遺伝子
KR20130038914A (ko) 글루코스 탈수소효소
EP4067483A1 (fr) Glucose déshydrogénase mutante présentant une meilleure stabilité thermique et utilisation correspondante
JP2010054503A (ja) グルコース脱水素酵素を用いたグルコースの電気化学測定法
JP4872033B1 (ja) 抗原を検出する方法
JPWO2017183717A1 (ja) ＨｂＡ１ｃデヒドロゲナーゼ
JP6623406B2 (ja) 結合剤
WO2017026505A1 (fr) Agent de liaison
JP2017037015A (ja) 分子識別素子固定化電極
JP5896375B2 (ja) 改変型グルコース脱水素酵素遺伝子
JP5592440B2 (ja) グルコース脱水素酵素
WO2017008816A1 (fr) Électrode enzymatique pour la régénération de formes oxydées et/ou réduites de nucléotides pyridiniques
JP6940149B2 (ja) フラビン結合型グルコース脱水素酵素
JP2021078427A (ja) Ｄ−乳酸脱水素酵素変異体、ｄ−乳酸脱水素酵素変異体を備えたバイオ燃料電池、及び、バイオセンサ
US20220306996A1 (en) Mutant Glucose Dehydrogenase Having Improved Thermal Stability, and Use Thereof
US20210292804A1 (en) Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof
Lee et al. Protocol for construction and characterization of direct electron transfer-based enzyme-electrode using gold binding peptide as molecular binder
US20200399671A1 (en) Fusion protein of flavin adenine dinucleotide-glucose dehydrogenase and cytochrome molecule
JP2016116470A (ja) 変異型ピロロキノリンキノン依存性グルコース脱水素酵素、変異型ピロロキノリンキノン依存性グルコース脱水素酵素をコードする単離核酸分子、変異型ピロロキノリンキノン依存性グルコース脱水素酵素を固定した酵素電極、酵素電極を備えたバイオ電池、及びバイオセンサー
JP5939641B2 (ja) グルコース脱水素酵素

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16835202

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16835202

Country of ref document: EP

Kind code of ref document: A1