WO2004072283A1 - Antifreeze protein containing ice nucleating protein sequence - Google Patents

Antifreeze protein containing ice nucleating protein sequence Download PDF

Info

Publication number
WO2004072283A1
WO2004072283A1 PCT/JP2004/001728 JP2004001728W WO2004072283A1 WO 2004072283 A1 WO2004072283 A1 WO 2004072283A1 JP 2004001728 W JP2004001728 W JP 2004001728W WO 2004072283 A1 WO2004072283 A1 WO 2004072283A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
peptide
antifreeze
amino acid
acid sequence
Prior art date
Application number
PCT/JP2004/001728
Other languages
French (fr)
Japanese (ja)
Inventor
Yoshihiro Kobashigawa
Satoru Ohgiya
Ai Miura
Sakae Tsuda
Original Assignee
National Institute Of Advanced Industrial Science And Technology
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
Application filed by National Institute Of Advanced Industrial Science And Technology filed Critical National Institute Of Advanced Industrial Science And Technology
Priority to JP2005505033A priority Critical patent/JP4231928B2/en
Publication of WO2004072283A1 publication Critical patent/WO2004072283A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K3/00—Materials not provided for elsewhere
    • C09K3/18—Materials not provided for elsewhere for application to surfaces to minimize adherence of ice, mist or water thereto; Thawing or antifreeze materials for application to surfaces
    • C09K3/185—Thawing materials

Definitions

  • the present invention relates to an antifreeze protein or peptide which is designed and partially prepared by using a part of a repetitive sequence of an ice nucleus protein, and further relates to a method for producing the protein or peptide.
  • the present invention relates to a use, a DNA encoding the protein or the peptide, a vector containing the DNA, and a transformant containing the vector.
  • Antifreeze proteins exhibit properties such as 1) temperature hysteresis, 2) eternal recrystallization inhibition, and 3) eternal crystal shape control. It has been proposed to be added to ice cream and used as a cryopreservative for cells and organs (Greffith M and Ewart KV, 1995. Biotechnology Advance 13: 375-402). It is also expected to be an effective additive that can eliminate clogging of piping systems due to ice recrystallization in cold heat supply systems or cold heat storage that uses ice slurry.
  • antifreeze proteins derived from animals and plants has been used to maintain the quality of frozen foods such as ice cream, improve the tolerance of blood, cells, eggs, spermatozoa, and transplanted organs to be frozen, and use them in cold supply systems or cold storage. Applications are being attempted.
  • antifreeze proteins are found only in specific fish species inhabiting polar regions, and are expressed in large quantities only during the winter season, so they are stable throughout the year. And it cannot be mass-produced.
  • the recovery of antifreeze protein from fish also has problems such as variations in quality between mouth and mouth due to differences in the expression level of antifreeze protein depending on when and where the fish was caught, and on individuals.
  • antifreeze proteins have been found in many organisms such as fish and fungi. Among them, antifreeze proteins that have been studied extensively include those derived from fish that inhabit the Arctic and Antarctic waters.
  • antifreeze proteins such as 1) temperature hysteresis, 2) inhibition of ice recrystallization, and 3) control of ice crystal shape, are due to the fact that antifreeze proteins bind to ice It is thought to be due to the inhibition of the binding of nuclei to the growth of ice nuclei.
  • Antifreeze proteins derived from fish are classified into four types according to their structure.
  • Type I antifreeze protein is an alpha helix type protein that contains many Ala residues and Thr and Asp residues are equally spaced.
  • ⁇ type antifreeze protein is a C-type lectin-like protein containing disulfide bonds.
  • Type III antifreeze proteins are globular proteins with characteristic structural motifs.
  • Type IV antifreeze protein contains many Gin residues and is an unknown protein.
  • an antifreeze protein from Ligustrum beetle Liou Y et al., 2001. Nature, 406: 322-324; Graether SP et al., 2001, Nature. 406: 325-328
  • Ice-nucleated proteins are proteins that promote ice crystal growth, while antifreeze proteins inhibit ice crystal growth. Therefore, it is thought that both the ice core protein and the antifreeze protein have a structure that can bind to a frozen water molecule.
  • An ice nucleus protein derived from Pseudomonas syringae which is a type of ice bacterium, is a membrane protein of approximately 1200 residues, with an N-terminal region (; ⁇ 19 kDa), a central region ( ⁇ 94 kDa), and a C-terminal region. ( ⁇ 7 kDa).
  • the central region is composed of a characteristic repeating amino acid sequence, which is thought to be the site involved in binding to ice crystals (Abe K et al., 1989. FEBS Lett., 258: 297-300; Green RL et al., 215, Mol. Gen. Genet., 215: 165-172; Corotto LV et al., 1986, EMB0 J., 5: 231-236). Therefore, when the present inventors artificially synthesize a peptide having a size similar to that of an antifreeze protein by referring to the sequence of the central region of the ice nucleus protein, the peptide becomes It is thought to have such an ice crystal growth suppression function.
  • the present invention is intended to solve such a conventional problem.
  • a protein or a peptide which can be stably produced throughout the year by gene expression, chemical synthesis, etc. and has an antifreeze ability. Is newly provided.
  • the present inventors have found that a portion consisting of an amino acid sequence of -Thr-Xxx-Thr- is present on one side of an antifreeze protein having a beta-1 'sheet structure, and further, the hydroxyl group of this Thr is Based on the finding that they are regularly arranged at positions where hydrogen bonds can be formed with the crystal plane, we have examined whether there is any other protein containing the -Thr-Xxx-Thr- sequence. As a result, they found that the -Thr-Xxx-Thr- sequence exists in the central region of the ice nucleus protein. The peptide containing the sequence of this region was synthesized by a genetic engineering technique, and it was confirmed that the obtained peptide actually had an antifreeze function, thereby completing the present invention. is there.
  • the present invention is as follows.
  • a protein or peptide having an antifreeze function comprising an amino acid sequence represented by SEQ ID NO: 12.
  • a protein or a peptide having an antifreeze function comprising an amino acid sequence represented by SEQ ID NO: 13.
  • (B) a protein comprising an amino acid sequence containing an amino acid sequence in which one or several amino acid residues have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4, and having an antifreeze function Or peptides.
  • a protein or a peptide having an antifreeze function comprising an amino acid sequence containing a repeated amino acid sequence in an ice core protein.
  • the protein or peptide according to any one of (1) to (5) is encoded. DNA fragment.
  • a method comprising culturing the transformant according to (10) or (11) to produce a protein or peptide having an antifreeze function, and collecting the protein or peptide. For producing a protein or peptide having an antifreeze function.
  • An ice recrystallization inhibitor or a freezing point depressant comprising the protein or peptide according to any one of (1) to (5).
  • a peptide is a substance in which two or more amino acids are linked by a peptide bond (“Physical and Chemical Dictionary” (5th edition), published by Iwanami Shoten, p. March 20)) Protein is also a kind of peptide.
  • a peptide as small as about 30 residues is referred to as a peptide, and a peptide larger than that is referred to as a protein.
  • proteins having antifreeze ability are called antifreeze proteins, and peptides having antifreeze ability are called antifreeze peptides to distinguish them. For simplicity, these are collectively referred to as “antifreeze proteins”.
  • ice crystals first grow into flat hexagonal plates when ice nuclei appear in an aqueous solution. Growth in the direction perpendicular to the plate-like plane is about 100 times slower than growth in the direction of the plate-like plane.
  • AFP antifreeze protein
  • the plate-like body formed first is used as the base surface, and the growth direction is perpendicular to the base surface.
  • smaller platelets are stacked one after the other, eventually resulting in a bivilamid-type ice crystal with two viramids as shown in Fig. 1. It grows up.
  • the sample liquid contains bipyramid ice crystals and crystallography as shown in Fig. 1.
  • ice crystals called hexagonal bipyramids are observed under a microscope.
  • Such bipyramidal ice crystals are formed as a result of the ability of AFP to specifically bind to 12 ice planes on the ice crystal.
  • This is macroscopically observed as a non-freezing phenomenon (antifreeze activity) of the specimen.
  • This phenomenon can be quantified as the freezing point depression or temperature hysteresis of the sample liquid by using an osmometer (osmometer).
  • the antifreeze activity evaluation method by observing the viviramidal ice crystal is observed if AFP is present.
  • the simplest and quickest way to evaluate the presence of AFP in a sample ie, an aqueous solution of the protein of interest, is to observe the viviramid ice crystals in the sample solution.
  • FIG. 2 shows the structural model of the beta-sheet antifreeze protein derived from the locust beetle, Jacuzzi beetle, and Figure 3 shows the ice crystal surface binding model of the protein.
  • An ice crystal binding site -Thr-Xxx-Thr- exists on the surface of these molecules. This surface has a structure complementary to the surface of the ice crystal, and the hydroxyl group of Thr forms a hydrogen bond with oxygen atoms regularly arranged on the ice crystal surface.
  • FIG. 4 and SEQ ID NO: 14 show the amino acid sequence of the ice core protein of Pseudomonas syringae, a kind of gram-negative bacteria.
  • the ice core protein is a membrane protein having a molecular weight of about 120 kDa, and is composed of three regions: an N-terminal region ( ⁇ 19 kDa), a central region ( ⁇ 94 kDa), and a C-terminal region ( ⁇ 7 kDa).
  • N-terminal region ⁇ 19 kDa
  • central region ⁇ 94 kDa
  • C-terminal region ⁇ 7 kDa.
  • the amino acid sequence represented by SEQ ID NO: 12 in this sequence includes -Thr-Xxx-Thr-, which is an amino acid sequence involved in ice crystal binding of an antifreeze protein derived from P. chinensis (Double underline in Fig. 4). Based on the above findings, the present inventor has found that the amino acid sequence portion including -Thr-Xxx-Thr- in the central region of the ice core protein has an I predicted that it could be combined.
  • the protein of the present invention has at least the amino acid sequence represented by SEQ ID NO: 12 or the amino acid sequence represented by SEQ ID NO: 13, and includes a protein or peptide having an antifreeze function, Further, it includes a protein or peptide in which the amino acid sequence represented by SEQ ID NO: 13 is repeated a plurality of times, and a protein comprising the above 96 amino acid residues represented by SEQ ID NO: 4 in the sequence listing. Also. Even if one or several amino acid residues are deleted, substituted, or added in the amino acid sequence of the protein consisting of 96 amino acid residues, the protein having the antifreeze function is not subject to this invention. Included in the invention.
  • 1 to 7, preferably 1 to 5, and more preferably 1 to 2 amino acids of the amino acid sequence represented by SEQ ID NO: 4 may be deleted, or the amino acid sequence represented by SEQ ID NO: 4 may be deleted.
  • 1 to 7, preferably 1 to 5, more preferably 1 to 2 amino acids of the amino acid sequence may be added, or 1 to 7 of the amino acid sequence represented by SEQ ID NO: 4.
  • Seven, preferably one to five, more preferably one to two amino acids may be replaced by another amino acid.
  • FIG. 5 and SEQ ID NO: 15 and FIG. 6 and SEQ ID NO: 16 show the sequences of the antifreeze protein derived from Pseudococcidae and the INP96, and in both proteins, -Thr-Xxx-Thr- It can be seen that the sequence is repeated four times. Also, recent studies have shown that in the connection between ice crystals and antifreeze protein, the complementarity of the shape of the ice crystal surface and the surface of the antifreeze protein rather than the hydrogen bond between the antifreeze protein and oxygen atoms on the ice crystal surface.
  • the protein or peptide of the present invention may be synthesized using a peptide synthesizer, or a water core protein or its C-terminal domain peptide or an acid, It can also be produced by limited partial decomposition using a hydrolysis reagent. In the case of performing this partial decomposition, a peptide having no antifreeze function may be produced at the same time as a protein having an antifreeze function, but it has a length of at least a certain length.
  • the protein having the amino acid sequence represented by SEQ ID NO: 12 can be regarded as a protein having an antifreeze function.
  • the protein may be purified by a conventional method, but may contain other proteins or peptides, and can exhibit an antifreeze function without purification.
  • the obtained protein or the like has an antifreeze function is determined, as described above, by the presence or absence of the aforementioned pyramid-shaped ice crystals at the freezing temperature in an aqueous solution of the protein or the like.
  • the screen can be screened easily and quickly.
  • the antifreeze activity of INP96 can be confirmed by observing the recrystallization inhibitory activity with a microscope or by measuring the degree of freezing point depression with a water point depression osmometer.
  • the antifreeze protein or peptide of the present invention can be used as an ice recrystallization inhibitor or a freezing point depressant. As a specific application, for example, it can be used to maintain its quality by being mixed into ice cream or frozen food. Furthermore, in recent years, a cold heat supply system or a cold heat storage system using an ice slurry having a large energy density as a heat medium has been proposed. Since antifreeze proteins effectively prevent recrystallization of ice, they can be a promising tool to solve this problem. In addition, long-term storage of cells such as eggs and sperm at low temperatures can be expected as an applied technology.
  • Figure 1 is a schematic diagram of a viviramid ice crystal.
  • FIG. 2 is a diagram showing a three-dimensional structural model of an antifreeze protein derived from the locust beetle, Chinoen beetle.
  • FIG. 3 is a diagram showing an ice crystal surface binding model of an antifreeze protein derived from the cynomolgus duck beetle.
  • FIG. 4 is a view showing the N-terminal region of the ice core protein inaZ, the amino acid sequences of the central region and the C-terminal region, and the INP96 portion (underlined portion) in the central region.
  • FIG. 5 shows the amino acid sequence of INF96.
  • the underlined part indicates the part of the TXT array.
  • FIG. 6 is a diagram showing the amino acid sequence of an antifreeze protein derived from the locust beetle, Dermatophagoides farinae. The underlined part indicates the part of the TXT array.
  • FIG. 7 shows the DNA sequence of INP96.
  • FIG. 8 is a chromatogram showing the elution pattern of INP96 on a chitin column.
  • FIG. 9 is an SDS-PAGE of the elution fraction of INP96 on a chitin column.
  • Figure 10 is a photograph of a piviramid-type ice crystal observed for a 9 mg / ml aqueous solution of INP96.
  • FIG. 11 is a graph showing the results of measuring the concentration dependence of the freezing point of chicken egg white lysozyme and INP96.
  • INP96aalf SEQ ID NO: 6
  • INP96aalr SEQ ID NO: 7
  • INP96aa2f SEQ ID NO: 8
  • INP96aa2r SEQ ID NO: 9
  • Second step 9 Cycle at 4 ° C for 30 seconds (denaturation), ramp down to 65 ° C over 1 minute, cycle at 65 ° C for 30 seconds (annealing), 72 ° C for 1.5 minutes (extension) 35 times, Third step: PCR reaction was performed at 72 ° C for 5 minutes.
  • the double-stranded DNAs produced by ligation were ligated to pT7Blue T-vector (Novagen), and Escherichia coli DH5a (Biotechnology Engineering Research Division, National Institute of Advanced Industrial Science and Technology) ) was transformed.
  • the transformant was spread on an LB plate containing 1 mM IPTG, 0.02% X-Gal, 100 mg / ml ampicillin, and cultured at 37 ° C for about 20 hours. Eight of the white colonies that emerged were cultured in 3 ml of LB medium containing lOOmg / ml ampicillin in a test tube at 37 ° C at 37 ° C.
  • the sequences of the primers used in the second PCR reaction were INP96f2 (SEQ ID NO: 10) and INP961-2 (SEQ ID NO: 11).
  • KOD polymerase KOD polymerase
  • Toyobo Co., Ltd. which is known to have a low error frequency, was used in order to minimize the possibility of errors occurring during the PCR reaction.
  • the PCR was performed using the above plasmid solution diluted 5000 times, dNTP 200 mM, magnesium chloride 1 mM, INP96f2, INP96r2 300 nM each, buffer (IX) and K0D-plus- attached to K0D-Plus- (KOD-Plus-).
  • the reaction was performed with 50 ml of a reaction solution containing 1 U of a polymerase (KOD-Plus-polymerase).
  • the recovered DNA was ligated to a pZEr02 vector (Invtrogen) cut in advance with Eco RV, and Escherichia coli DH5a was transformed using the vector.
  • Transformants were plated on LS-LB (1% Trypton, 0.5% Yeast Extract, 0.5% sodium chloride) plates containing 1 mM IPTG and 50 mg / ml kanamycin. After culturing at 37 ° C for about 20 hours, 8 of the emerged colonies were cultured in 3 ml of LS-LB medium containing 50 mg / ml kanamycin at 37 ° C overnight in a test tube.
  • Plasmids are prepared using the Plasmid MiniPreb Kit (QuantumPrep Plasraid MiniPrep kit), and three transformants carrying the plasmid that is thought to contain the target DNA are identified based on the restriction enzyme cleavage pattern. did. For these, the base sequences were confirmed using Big Dye, Terminator, Cycle, Sequencing, Ready, and Reaction 'kit ver. 2.0. As a result, it was possible to obtain the DNA plasmid that is ⁇ (P ZEr02INP96) having the nucleotide sequence of interest shown in FIG. Next, the transformant holding the plasmid was cultured in a 50-ml culture solution, and a plasmid was prepared using a Quantumpreb-plasmid-mieprep kit (Bio-Rad).
  • IMPACT-CN system New England Biolabs
  • a target protein is produced as a fusion protein with a chitin-binding protein and a protease, intin.
  • the expression vector used in the system encodes, in advance, a nucleotide sequence encoding a chitin-binding domain that facilitates purification of the expressed protein, and an autolyzing protease-tin for cutting off the target protein.
  • the base sequence has been incorporated.
  • pZEr02INP96 was digested with NdeI and EcoRI, and the DNA encoding about 300 bp of INP96 was separated and recovered by agarose electrophoresis using nucleic acid GTG agarose.
  • the expression vector PTYB12 contained in the system was cut with NdeI and EcoRI. Both were ligated using a Takara DNA Ligation kit (Takara) according to the attached protocol to transform Escherichia coli DH5a. The transformant was spread on an LB plate containing 100 mg / ml ampicillin, and cultured at 37 ° C for approximately 18 hours.
  • Appear 18 colonies were cultured in 3 ml of LB medium containing lOOmg / ml ampicillin at 37 ° C in a test tube, and then prepared with Quantum Preb, Plasmid, Mini Preb Kit and prepared. From the pattern of cleavage by the restriction enzyme, 10 transformants carrying the plasmid likely to contain the target DNA were identified. One of these transformants was cultured in a 50 ml culture solution, and a plasmid was prepared using Quantumpreb 'Plasmid. Minipreb kit. In addition, the base sequence was confirmed using Big Dye Terminator Cycle 'Sequen Sync', Ready, Reaction, Kit ver. 2.0. As a result, a plasmid (pTYB12INP96) in which the DNA having the target nucleotide sequence shown in FIG. 7 was inserted into pTYB12 was obtained.
  • INP96 was expressed in Escherichia coli as a fusion protein with chitin-binding domain and tintin.
  • Escherichia coli ER2566 (DE3) (New England Biolab) was transformed with plasmid pTYB12INP96. Since an ampicillin resistance gene was introduced into plasmid pYT12INP96, transformants were selected by spreading Escherichia coli on LB agar medium containing ampicillin and incubating at -37 ° C. One of the formed colonies was inoculated in 15 ml of LB medium containing 100 ug / ml of ampicillin, and cultured at 28 ° C.
  • This culture was subcultured into a medium 1.51 containing 100 g / ml of ampicillin and 0.5 mM isopropyl-D (-)-thiogalactobyranoside, and further cultured at 28 ° C.
  • the culture was centrifuged at 3600 X g at 4 ° C for 15 minutes to collect the cells.
  • the cells 20 mM Tris - was suspended in HCl buffer I ImM Echirenjiamin tetraacetate Ninatori um (P H 8. 0), and ultrasonic Yabu ⁇ in ice. This was centrifuged at 11,900 X g at 4 ° C for 60 minutes to separate a soluble fraction and an insoluble fraction.
  • INP96 in the form of a fusion protein with chitin-binding domain and intin extracted from the soluble fraction was placed on a chitin column buffered with 20 mM Tris-HCl buffer I ImM ninatrium ethylenediaminetetraacetate / 0.5 M NaCl aqueous solution (pH 8.0). Adsorbed.
  • the purified INP96 was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (ATT0).
  • Broad range SDS-PAGE standard (BIO-RAD) was run simultaneously for molecular weight measurement (sample buffer was 0.065 M Tris-HCl buffer (pH 6.8) / 2 % Sodium dodecylsulfate I 10% sucrose / 5% ⁇ -mercaptoethanol I 0.001% bromophenol blue concentrated gel 0.5 0 Tris-HCl buffer ( ⁇ 6.8) 0 75 ml, 0.45 ml of 30% acrylamide Ibis (37.5: 1) mixture, 0.12 ml of 10% sodium dodecinole sulfate and 1.78 ml of distilled water are mixed and prepared.
  • the gel is 1.5 M Tris-HCl buffer (pH 8.8) 2.25 ml, 30% acrylamide Ibis (37.5: 1) mixture 4, 5 ml, 10% sodium dodecyl sulfate 0
  • the mixture was prepared by mixing 36 ml of distilled water and 1.89 ml of distilled water
  • the electrophoresis buffer was 3.03 g of tris (hydroxymethyl) amino methane, 14.4 g of glycine, and the like.
  • a solution of sodium dodecyl sulfate lg in a total of 11 distilled water was used for gel staining: methanol 20 ml, ammonium sulfate 12.5 g, phosphoric acid 2.5 ml, 0.04% Coomassie brilliant Performed overnight using a staining solution prepared by dissolving Blue G-250 in 100 ml of distilled water, and destained with distilled water From Fig. 9, it can be confirmed that INP96 having a molecular weight of 11 KDa was highly purified. .
  • the temperature in the cooling box in which the sample solution is set is controlled with an error of +/- 0.1 ° C by a Linkam LK600 temperature controller (Linkam LK600 temperature controller).
  • Linkam LK600 temperature controller Linkam LK600 temperature controller
  • the temperature in the cooling box was decreased from 0.2 ° C per second to -22 ° C by the temperature controller. Approximately -14 ° C to -22. Somewhere in the temperature between C, the whole sample freezes. After freezing, raise the temperature inside the cooling box at 0.2 ° C per second, stop rising at zero degree, and keep it at -3 ° C for about 1 to 10 seconds. After passing through a single ice crystal state, countable ice crystals were observed floating in the water.
  • the control chemical hen egg white lysozyme
  • INP96 shows a non-linear decrease in freezing point up to 0.12 ° C, which is observed only when the antifreeze protein in the aqueous solution specifically interacts with the ice crystal surface. .
  • This experiment confirmed that INP96 exhibited antifreeze protein-like freezing point lowering activity.
  • the present invention is to design, create and supply antifreeze proteins based on the structure of ice core proteins.
  • antifreeze proteins or antifreeze peptides can be easily and massively supplied by producing them by peptide synthesis, genetic engineering, or partial decomposition of ice nucleus proteins.
  • SEQ ID NO: 5 DNA sequence encoding INP96aa
  • SEQ ID NO: 6 primer for constructing DNA sequence encoding INP96aalf
  • SEQ ID NO: 7 primer for constructing DNA sequence encoding INP96aalr
  • SEQ ID NO: 8 Primer for constructing a DNA sequence encoding INP96aa2f
  • SEQ ID NO: 9 Primer for constructing DNA sequence encoding INP96aa2r
  • SEQ ID NO: 10 Primer for constructing DNA sequence encoding INP96f2
  • SEQ ID NO: 11 Primer for constructing DNA sequence encoding INP96r2
  • SEQ ID NO: 12: Xaa represents any amino acid residue
  • SEQ ID NO: 13: Xaa represents any amino acid residue
  • SEQ ID NO: 15 Synthetic amino acid sequence
  • SEQ ID NO: 16 Synthetic amino acid sequence

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Combustion & Propulsion (AREA)
  • Materials Engineering (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

A method of obtaining a peptide having an antifreeze function which comprises designing an antifreeze protein or peptide with the use a part of a repeated sequence of an ice nucleating protein and then effecting, based on the thus obtained result, gene expression, chemical synthesis or partial digestion of disrupted ice nucleating microbial cells.

Description

明 細 書 氷核蛋白質の配列を含む不凍蛋白質 技術分野  Description Antifreeze protein containing the sequence of ice core protein
本発明は、 氷核蛋白質の繰り返し配列の一部を利用して設計し、 新たに調製し 'て得られた不凍蛋白質またはぺプチドに関するもので、 さらに該蛋白質またはべ プチドの製造方法、 その用途、 該蛋白質またはペプチドをコードする DNA、 該 DNA を含むベクター、 およぴ該ベクターを含む形質転換体に関する。 背景技術  The present invention relates to an antifreeze protein or peptide which is designed and partially prepared by using a part of a repetitive sequence of an ice nucleus protein, and further relates to a method for producing the protein or peptide. The present invention relates to a use, a DNA encoding the protein or the peptide, a vector containing the DNA, and a transformant containing the vector. Background art
不凍蛋白質は、 1) 温度ヒステリシス、 2) 永の再結晶阻害、 3) 永結晶形状の 制御等の特性を示すため、 保冷により周囲の水分子が付着再結晶することで風味 や味が損なわれるアイスクリームへの添加や、 細胞や臓器の冷凍保存剤に用いる こ とが提案されている ( Greffith M and Ewart KV, 1995. Biotechnology Advance 13： 375-402) 。 また、 氷スラリーを使用する冷熱供給システムまたは 冷熱蓄熱等において、 氷の再結晶による配管系の閉塞を解消し得る有効な添加剤 としても期待されている。 これまでは、 動植物由来の不凍蛋白質を用いてアイス クリーム等の冷凍食品の品質保持、 血液や細胞、 卵子や精子、 移植臓器の冷凍保 存耐性の向上、 冷熱供給システムまたは冷熱蓄熱等への応用が試みられている。  Antifreeze proteins exhibit properties such as 1) temperature hysteresis, 2) eternal recrystallization inhibition, and 3) eternal crystal shape control. It has been proposed to be added to ice cream and used as a cryopreservative for cells and organs (Greffith M and Ewart KV, 1995. Biotechnology Advance 13: 375-402). It is also expected to be an effective additive that can eliminate clogging of piping systems due to ice recrystallization in cold heat supply systems or cold heat storage that uses ice slurry. Until now, the use of antifreeze proteins derived from animals and plants has been used to maintain the quality of frozen foods such as ice cream, improve the tolerance of blood, cells, eggs, spermatozoa, and transplanted organs to be frozen, and use them in cold supply systems or cold storage. Applications are being attempted.
しカゝし、 現状では不凍蛋白質類は、 極地方に生息する特定の魚種にしか見いだ されておらず、 また、 冬季間にしか大量には発現されないので、 年間を通して安 定にかつ大量に生産することができない。 また、 魚類からの不凍蛋白質の回収に おいては漁獲した時期および場所、 ならびに個体により不凍蛋白質の発現量が異 なるために口ット間の品質のばらつきを生じる等の問題がある。  However, at present, antifreeze proteins are found only in specific fish species inhabiting polar regions, and are expressed in large quantities only during the winter season, so they are stable throughout the year. And it cannot be mass-produced. In addition, the recovery of antifreeze protein from fish also has problems such as variations in quality between mouth and mouth due to differences in the expression level of antifreeze protein depending on when and where the fish was caught, and on individuals.
前述のとおり不凍蛋白質は魚や菌類など多くの生物から発見されている。 これ らの中でも特に幅広く研究が行われている不凍蛋白質として、 北極海や南極海に 生息する魚類由来の不凍蛋白質が挙げられる。  As described above, antifreeze proteins have been found in many organisms such as fish and fungi. Among them, antifreeze proteins that have been studied extensively include those derived from fish that inhabit the Arctic and Antarctic waters.
不凍蛋白質における、 1) 温度ヒステリシス、 2) 氷の再結晶阻害、 3) 氷結晶 形状の制御等の特性は、 不凍蛋白質が氷表面に結合することにより新たな水分子 の結合を阻害し氷核の成長を妨げることによるものと考えられている。 そして、 魚類由来の不凍蛋白質はその構造により 4種類に分類されている。 I 型の不凍蛋 白質は Ala残基を多く含み Thr残基と Asp残基が等間隔で配置されたアルファ · へ リ ックス型の蛋白質である。 Π 型の不凍蛋白質はジスルフィ ド結合を含み C型 - レクチン様の蛋白質である。 III 型の不凍蛋白質は特徴的な構造モチーフから成 る球状蛋白質である。 IV型の不凍蛋白質は Gin残基を多く含み立体構造未知の蛋 白質である。 また、 近年明らかにされたチヤイロコメノゴミムシダマシ由来の不 凍蛋白質 (Liou Y et al. , 2001. Nature, 406： 322-324； Graether SP et al. , 2001， Nature. 406： 325 - 328)は、 ペータ ' シート構造からなる蛋白質である。 このように、 不凍蛋白質の構造にはバリエーションがあり、 このことが不凍蛋白 質の氷結晶結合とその成長抑制メカ二ズムの理解を困難にしている。 The characteristics of antifreeze proteins, such as 1) temperature hysteresis, 2) inhibition of ice recrystallization, and 3) control of ice crystal shape, are due to the fact that antifreeze proteins bind to ice It is thought to be due to the inhibition of the binding of nuclei to the growth of ice nuclei. Antifreeze proteins derived from fish are classified into four types according to their structure. Type I antifreeze protein is an alpha helix type protein that contains many Ala residues and Thr and Asp residues are equally spaced.不 type antifreeze protein is a C-type lectin-like protein containing disulfide bonds. Type III antifreeze proteins are globular proteins with characteristic structural motifs. Type IV antifreeze protein contains many Gin residues and is an unknown protein. In addition, an antifreeze protein from Ligustrum beetle (Liou Y et al., 2001. Nature, 406: 322-324; Graether SP et al., 2001, Nature. 406: 325-328), which was recently identified, was , Peter 'is a protein consisting of a sheet structure. Thus, there are variations in the structure of antifreeze proteins, which makes it difficult to understand the binding of ice crystals to antifreeze proteins and their growth-inhibitory mechanisms.
不凍蛋白質が氷の結晶成長を抑制するのに対して、 氷核蛋白質は氷の結晶成長 を促進する蛋白質である。 このため、 氷核蛋白質と不凍蛋白質はともに凍結状態 にある水分子と結合できる構造を有すると考えられている。 氷核菌の一種である シュゥドモナス · シリンガエ (Pseudomonas syringae) 由来の氷核蛋白質はおよ そ 1200残基の膜蛋白質であり、 N末端領域 (；〜 19kDa)、 中心領域（〜94kDa)、 C末端 領域（〜7kDa)の 3つの領域から構成される。 これら 3つの領域のうち、 中心領域 は特徴的な繰り返しァミノ酸配列から構成されてぉり、 この配列が氷結晶との結 合に関わる部位と考えられている （Abe K et al. , 1989. FEBS Lett. , 258： 297-300； Green RL et al. , 215, Mol. Gen. Genet. , 215： 165 - 172 ; Corotto LV et al.， 1986, EMB0 J. , 5： 231-236) 。 従って、 本発明者らは、 この氷核蛋 白質の中心領域の配列を参考にして不凍蛋白質と同程度の大きさをもつペプチド を人工的に合成した場合、 そのペプチドは、 不凍蛋白質のような氷結晶成長抑制 機能を有すると考えた。  Ice-nucleated proteins are proteins that promote ice crystal growth, while antifreeze proteins inhibit ice crystal growth. Therefore, it is thought that both the ice core protein and the antifreeze protein have a structure that can bind to a frozen water molecule. An ice nucleus protein derived from Pseudomonas syringae, which is a type of ice bacterium, is a membrane protein of approximately 1200 residues, with an N-terminal region (; ~ 19 kDa), a central region (~ 94 kDa), and a C-terminal region. (~ 7 kDa). Of these three regions, the central region is composed of a characteristic repeating amino acid sequence, which is thought to be the site involved in binding to ice crystals (Abe K et al., 1989. FEBS Lett., 258: 297-300; Green RL et al., 215, Mol. Gen. Genet., 215: 165-172; Corotto LV et al., 1986, EMB0 J., 5: 231-236). Therefore, when the present inventors artificially synthesize a peptide having a size similar to that of an antifreeze protein by referring to the sequence of the central region of the ice nucleus protein, the peptide becomes It is thought to have such an ice crystal growth suppression function.
これまで、 主に植物、 魚類由来の不凍蛋白質を用いてアイスクリーム等の冷凍 食品の品質保持、 細胞の冷凍保存耐性の向上が試みられてきたが実用化には至つ ていない。 また、 冷熱供給システム、 冷熱蓄熱等を含め、 その期待される有効性 の高さにもかかわらず実用化に結びつかない主な理由は、 不凍蛋白質が北極や南 極に生息する魚類の血液等から得られる極めて高価な抽出物であること、 および これらを安定的に生産する技術が存在しないことである。 発明の開示 Until now, attempts have been made to maintain the quality of frozen foods, such as ice cream, and to improve the resistance to frozen storage of cells using mainly antifreeze proteins derived from plants and fish, but this has not been put to practical use. Despite its expected high effectiveness, including the cold heat supply system and cold heat storage, the main reason why it cannot be put to practical use is that antifreeze proteins are found in fish blood inhabiting the Arctic and Antarctic. And that there is no technology for producing them stably. Disclosure of the invention
本発明は、 このような従来の問題を解決しょうとするものであり、 遺伝子発現、 化学合成などにより、 年間を通じて安定的に生産可能であって、 不凍能力を有す る蛋白質またはべプチド.を新たに提供するものである。  The present invention is intended to solve such a conventional problem.A protein or a peptide which can be stably produced throughout the year by gene expression, chemical synthesis, etc. and has an antifreeze ability. Is newly provided.
かかる状況において、 本発明者らは、 ベータ一 'シート構造から構成される不 凍蛋白質の片面に- Thr-Xxx- Thr -のアミノ酸配列からなる部分が存在し、 更にこ の Thrの水酸基が氷結晶面と水素結合を形成できる位置に規則的に配置されてい るという知見を基に、 -Thr- Xxx-Thr-の配列をふくむ蛋白質が他に存在するか否 かを検討してきた。 その結果、 - Thr- Xxx- Thr-の配列が氷核蛋白質の中心領域に 存在することを見出した。 この領域の配列を含むようなぺプチドを遣伝子工学的 手法により合成し、 得られたぺプチドが実際に不凍機能を有することを確認して、 本発明を完成させるに至ったものである。  Under such circumstances, the present inventors have found that a portion consisting of an amino acid sequence of -Thr-Xxx-Thr- is present on one side of an antifreeze protein having a beta-1 'sheet structure, and further, the hydroxyl group of this Thr is Based on the finding that they are regularly arranged at positions where hydrogen bonds can be formed with the crystal plane, we have examined whether there is any other protein containing the -Thr-Xxx-Thr- sequence. As a result, they found that the -Thr-Xxx-Thr- sequence exists in the central region of the ice nucleus protein. The peptide containing the sequence of this region was synthesized by a genetic engineering technique, and it was confirmed that the obtained peptide actually had an antifreeze function, thereby completing the present invention. is there.
すなわち、 本発明は、 以下のとおりのものである。  That is, the present invention is as follows.
( 1 ) 配列番号 1 2で表されるァミノ酸配列を含むことを特徴とする、 不凍機能 を有する蛋白質またはぺプチド。  (1) A protein or peptide having an antifreeze function, comprising an amino acid sequence represented by SEQ ID NO: 12.
( 2 ) 配列番号 1 3で表されるァミノ酸配列を含むことを特徴とする、 不凍機能 を有する蛋白質またはべプチド。  (2) A protein or a peptide having an antifreeze function, comprising an amino acid sequence represented by SEQ ID NO: 13.
( 3 ) 配列番号 1 2または 1 3で表されるアミノ配列が複数回繰り返されている アミノ酸配列を含む ( 2 ) に記載の蛋白質またはペプチド。  (3) The protein or peptide according to (2), which comprises an amino acid sequence in which the amino sequence represented by SEQ ID NO: 12 or 13 is repeated a plurality of times.
( 4 ) 次の（A)または（B)で表される蛋白質またはぺプチド。  (4) A protein or peptide represented by the following (A) or (B):
(A) 配列番号 4に記載のァミノ酸配列を有する蛋白質またはべプチド。  (A) a protein or a peptide having the amino acid sequence of SEQ ID NO: 4;
(B) 配列番号 4に記載のアミノ酸配列において、 1もしくは数個のアミノ酸残基 が欠失、 置換もしくは付加されたァミノ酸配列を含むァミノ酸配列からなり、 か つ、 不凍機能を有する蛋白質またはペプチド。  (B) a protein comprising an amino acid sequence containing an amino acid sequence in which one or several amino acid residues have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4, and having an antifreeze function Or peptides.
( 5 ) 氷核蛋白質における繰り返しアミノ酸配列を含むアミノ酸配列からなる不 凍機能を有する蛋白質またはべプチド。  (5) A protein or a peptide having an antifreeze function comprising an amino acid sequence containing a repeated amino acid sequence in an ice core protein.
( 6 ) 氷核蛋白質を化学的または生化学的に部分分解することを含む、 （1 ) 〜 ( 5 ) のいずれかに記載の蛋白質またはべプチドの製造方法。  (6) The method for producing a protein or a peptide according to any one of (1) to (5), which comprises partially decomposing an ice core protein chemically or biochemically.
( 7 ) ( 1 ) 〜 （5 ) のいずれか 1項に記載の蛋白質またはペプチドをコードす る DNA断片。 (7) The protein or peptide according to any one of (1) to (5) is encoded. DNA fragment.
(8) (7) に記載の DNA断片を含むベクター。  (8) A vector comprising the DNA fragment according to (7).
(9) ベクターが発現ベクターである （8) に記載のベクター。  (9) The vector according to (8), wherein the vector is an expression vector.
(1 0) (8) 〜 （9) のいずれか 1項に記載のベクターによって形質転換され た形質転換体。  (10) A transformant transformed by the vector according to any one of (8) to (9).
( 1 1) 宿主が大腸菌である （1 0) に記載の形質転換体。  (11) The transformant according to (10), wherein the host is Escherichia coli.
(1 2) (1 0) または （1 1) に記載の形質転換体を培養して不凍機能を有す る蛋白質またはべプチドを産生せしめ、 該蛋白質またはぺプチドを採取すること を特徴とする不凍機能を有する蛋白質またはべプチドの製造方法。  (12) A method comprising culturing the transformant according to (10) or (11) to produce a protein or peptide having an antifreeze function, and collecting the protein or peptide. For producing a protein or peptide having an antifreeze function.
( 1 3) 蛋白質またはペプチドをペプチド合成機により化学的に合成することを 特徴とする （1) 〜 （5) のいずれかに記載の蛋白質またはペプチドの製造方法。  (13) The method for producing a protein or peptide according to any one of (1) to (5), wherein the protein or peptide is chemically synthesized using a peptide synthesizer.
(1 4) (1) 〜 （5) のいずれかに記載の蛋白質またはペプチドを含むことを 特徴とする氷再結晶防止剤または凝固点降下剤。  (14) An ice recrystallization inhibitor or a freezing point depressant comprising the protein or peptide according to any one of (1) to (5).
(1 5) (1) 〜 (5) のいずれかに記載の蛋白質またはペプチドを含むことを 特徴とする氷スラリー。  (15) An ice slurry comprising the protein or peptide according to any one of (1) to (5).
以下、 本発明をさらに詳細に説明する。  Hereinafter, the present invention will be described in more detail.
まず、 不凍蛋白質または不凍ペプチドの機能について述べる。 ここで、 ぺプチ ドとは、 アミノ酸が 2個以上べプチド結合で連結した物質のことであり (岩波書 店発行 「理化学辞典」 (第 5版) 、 第 1254頁、 右襴（1998年 2月 20日）) 、 蛋白質 もペプチドの一種である。 ここでは、 慣例に従い、 30残基程度の小さいペプチド をペプチドと称し、 それ以上の大きなペプチドを蛋白質と称することにする。 す なわち、 不凍能力を有する蛋白質を不凍蛋白質と呼び、 不凍能力を有するぺプチ ドを不凍べプチド呼んで区別する。 また、 簡略化の為に、 これらをまとめて 「不 凍蛋白質類」 と呼称する。  First, the function of the antifreeze protein or antifreeze peptide will be described. Here, a peptide is a substance in which two or more amino acids are linked by a peptide bond (“Physical and Chemical Dictionary” (5th edition), published by Iwanami Shoten, p. March 20)) Protein is also a kind of peptide. Here, according to custom, a peptide as small as about 30 residues is referred to as a peptide, and a peptide larger than that is referred to as a protein. In other words, proteins having antifreeze ability are called antifreeze proteins, and peptides having antifreeze ability are called antifreeze peptides to distinguish them. For simplicity, these are collectively referred to as “antifreeze proteins”.
通常の場合、 氷結晶は、 水溶液中において氷核が表れると、 まず扁平な六角の 板状に成長する。 板状平面に対し垂直方向への成長は、 板状平面方向に対する成 長に比べ 100倍程度遅い。 これに対して、 不凍蛋白質 （AFP) が存在すると円盤平 面方向への氷晶の成長は阻止され、 最初に形成された板状体を基底面として、 こ の基底面に対し垂直方向に、 順次、 より小さい板状体が積み重ねられていき、 最 終的には図 1に示すようなビラミッドを二つ重ねたバイビラミッド型の氷晶にゆ つく りと成長していく。 Normally, ice crystals first grow into flat hexagonal plates when ice nuclei appear in an aqueous solution. Growth in the direction perpendicular to the plate-like plane is about 100 times slower than growth in the direction of the plate-like plane. On the other hand, when antifreeze protein (AFP) is present, the growth of ice crystals in the direction of the disk plane is prevented, and the plate-like body formed first is used as the base surface, and the growth direction is perpendicular to the base surface. In turn, smaller platelets are stacked one after the other, eventually resulting in a bivilamid-type ice crystal with two viramids as shown in Fig. 1. It grows up.
したがって、 注目する検体中に AFPが存在している場合に限り、 検体液を 0°C以 下にした場合、 その検体液中には図 1に示すようなバイピラミッド型氷結晶、 結 晶学的には六方両錐体とよばれる氷結晶が顕微鏡下に観測される。 AFPが有する 氷結晶上の 12枚の氷層平面に特異的に結合する能力の結果として、 このようなバ ィピラミッド型氷結晶が生成する。 このことが、 巨視的には、 検体の非凍結現象 (不凍活性） として観測される。 この現象は、 浸透圧計 （ォスモメーター） を用 いることにより検体液の凝固点降下または温度ヒステリシスとして定量化するこ ともできる。 凝固点降下の測定法を用いて不凍活性を評価する為には、 高純度の AFPの水溶液を得る必要がある。 これに対してバイビラミダル氷結晶観測による 不凍活性評価法は、 AFPさえ存在していれば観測される。 つまり、 検体すなわち 注目する蛋白質の水溶液中に AFPが存在するか否かを評価する為のもっとも簡便 で迅速な手法は、 検体液のバイビラミツド型氷結晶を観測することである。  Therefore, if the sample liquid is kept at 0 ° C or lower only when AFP is present in the sample of interest, the sample liquid contains bipyramid ice crystals and crystallography as shown in Fig. 1. Specifically, ice crystals called hexagonal bipyramids are observed under a microscope. Such bipyramidal ice crystals are formed as a result of the ability of AFP to specifically bind to 12 ice planes on the ice crystal. This is macroscopically observed as a non-freezing phenomenon (antifreeze activity) of the specimen. This phenomenon can be quantified as the freezing point depression or temperature hysteresis of the sample liquid by using an osmometer (osmometer). To evaluate antifreeze activity using the freezing point depression method, it is necessary to obtain a high-purity AFP aqueous solution. On the other hand, the antifreeze activity evaluation method by observing the viviramidal ice crystal is observed if AFP is present. In other words, the simplest and quickest way to evaluate the presence of AFP in a sample, ie, an aqueous solution of the protein of interest, is to observe the viviramid ice crystals in the sample solution.
チヤィ口コメノゴミムシダマシ由来のベータ一 ·シート型不凍蛋白質の構造モ デルを図 2に示し、 同蛋白質の氷結晶面結合モデルを図 3に示す。 これらの分子 表面には- Thr-Xxx- Thr-なる氷結晶結合部位が存在する。 この面は氷結晶の表面 と相補的な構造を有し、 Thrの水酸基は氷結晶面上に規則的に配列する酸素原子 との間で水素結合を形成する。 図 4およぴ配列番号 1 4に、 グラム陰性菌の一種 であるシユウドモナス ·シリンガエが有する氷核蛋白質のァミノ酸配列を示す。 該氷核蛋白質は分子量約 120kDaの膜蛋白質であり、 N末端領域 （〜19kDa) 、 中心 領域 （〜94kDa) 、 C末端領域 （〜7kDa) の 3つの領域から構成されている。 なお、 これらの各ァミノ酸配列は、 図 4のほか配列表の配列番号 1〜 3にも記載した (N末端領域一配列番号 1、 中心領域一配列番号 2、 C末端領域一配列番号 3 ) 。 図 4の中心領域のアミノ酸配列から明らかなように、 氷核蛋白質の中心領域に おいては、 48アミノ酸残基からなる同一または類似する配列が 20回繰り返されて いる繰り返しアミノ酸配列が存在する。 また、 この 48アミノ酸残基の繰り返しは、 16アミノ酸残基 （配列番号 1 3 ) の 3回の繰り返しから構成されている。 この配 列の中の配列番号 1 2で表されるアミノ酸配列は、 チヤイロコメノゴミムシダマ シ由来不凍蛋白質の氷結晶結合に関与するアミノ酸配列である- Thr - Xxx-Thr -を 含んでいる （図 4中の 2重下線部） 。 本発明者は、 上記の知見に基づき、 氷核蛋白質の中心領域の- Thr-Xxx- Thr -を 含むアミノ酸配列部分が、 チヤィ口コメノゴミムシダマシ由来の不凍蛋白質と同 様に、 氷結晶表面に結合できるのではないかと予測した。 この予測に基づき、 図 4中の一重下線部の 9 6アミノ酸残基からなる配列を有する蛋白質 （INP96；配 列番号 4に記载） を遺伝子組み換え手法を用いて合成した。 この 9 6残基の合成 蛋白質の水溶液中には、 図 1 0に示すように、 バイビラミッド型の永結晶が生成 した。 こうして、 氷核蛋白質の中心領域を構成する配列番号 1 2で表されるアミ ノ酸配列を有する蛋白質は、 不凍機能を有することが示された。 Figure 2 shows the structural model of the beta-sheet antifreeze protein derived from the locust beetle, Jacuzzi beetle, and Figure 3 shows the ice crystal surface binding model of the protein. An ice crystal binding site -Thr-Xxx-Thr- exists on the surface of these molecules. This surface has a structure complementary to the surface of the ice crystal, and the hydroxyl group of Thr forms a hydrogen bond with oxygen atoms regularly arranged on the ice crystal surface. FIG. 4 and SEQ ID NO: 14 show the amino acid sequence of the ice core protein of Pseudomonas syringae, a kind of gram-negative bacteria. The ice core protein is a membrane protein having a molecular weight of about 120 kDa, and is composed of three regions: an N-terminal region (末端 19 kDa), a central region (〜94 kDa), and a C-terminal region (〜7 kDa). Each of these amino acid sequences is also described in SEQ ID NOs: 1 to 3 in the sequence listing in addition to FIG. 4 (N-terminal region—SEQ ID NO: 1, central region—SEQ ID NO: 2, C-terminal region—SEQ ID NO: 3). . As is clear from the amino acid sequence of the central region in FIG. 4, in the central region of the ice core protein, there is a repeated amino acid sequence consisting of 48 amino acid residues and having the same or similar sequence repeated 20 times. In addition, the repetition of 48 amino acid residues is composed of three repetitions of 16 amino acid residues (SEQ ID NO: 13). The amino acid sequence represented by SEQ ID NO: 12 in this sequence includes -Thr-Xxx-Thr-, which is an amino acid sequence involved in ice crystal binding of an antifreeze protein derived from P. chinensis (Double underline in Fig. 4). Based on the above findings, the present inventor has found that the amino acid sequence portion including -Thr-Xxx-Thr- in the central region of the ice core protein has an I predicted that it could be combined. Based on this prediction, a protein (INP96; described in SEQ ID NO: 4) having a sequence consisting of 96 amino acid residues in single underline in FIG. 4 was synthesized using a genetic recombination technique. In the aqueous solution of the synthetic protein having 96 residues, as shown in FIG. 10, permanent bivilamid crystals were formed. Thus, it was shown that the protein having the amino acid sequence represented by SEQ ID NO: 12 constituting the central region of the ice core protein has an antifreeze function.
したがって、 本発明における蛋白質は、 少なくとも配列番号 1 2で表されるァ ミノ酸配列、 または配列番号 1 3で表されるアミノ酸配列を有し、 かつ不凍機能 を有する蛋白質またはペプチドを包含し、 さらには配列番号 1 3で表されるアミ ノ酸配列が複数回繰り返されている蛋白質またはぺプチド、 および配列表の配列 番号 4に示される上記 9 6アミノ酸残基からなる蛋白質を包含する。 また。 この 9 6了ミノ酸残基からなる蛋白質のァミノ酸配列において、 1もしくは数個のァ ミノ酸残基が欠失、 置換もしくは付加されたものであっても、 不凍機能を有する 蛋白質は本発明に含まれる。  Therefore, the protein of the present invention has at least the amino acid sequence represented by SEQ ID NO: 12 or the amino acid sequence represented by SEQ ID NO: 13, and includes a protein or peptide having an antifreeze function, Further, it includes a protein or peptide in which the amino acid sequence represented by SEQ ID NO: 13 is repeated a plurality of times, and a protein comprising the above 96 amino acid residues represented by SEQ ID NO: 4 in the sequence listing. Also. Even if one or several amino acid residues are deleted, substituted, or added in the amino acid sequence of the protein consisting of 96 amino acid residues, the protein having the antifreeze function is not subject to this invention. Included in the invention.
例えば、 配列番号 4で示されるァミノ酸配列の 1〜 7個、 好ましくは 1〜5個、 さ らに好ましくは 1〜2個のァミノ酸が欠失してもよく、 又は配列番号 4で表される 了ミノ酸配列の 1〜7個、 好ましくは 1〜5個、 さらに好ましくは 1〜2個のァミノ酸 が付加してもよく、 または、 配列番号 4で表されるアミノ酸配列の 1〜7個、 好ま しくは 1〜5個、 さらに好ましくは 1〜2個のアミノ酸が他のアミノ酸に置換されて いてもよい。  For example, 1 to 7, preferably 1 to 5, and more preferably 1 to 2 amino acids of the amino acid sequence represented by SEQ ID NO: 4 may be deleted, or the amino acid sequence represented by SEQ ID NO: 4 may be deleted. 1 to 7, preferably 1 to 5, more preferably 1 to 2 amino acids of the amino acid sequence may be added, or 1 to 7 of the amino acid sequence represented by SEQ ID NO: 4. Seven, preferably one to five, more preferably one to two amino acids may be replaced by another amino acid.
図 5と配列番号 1 5および図 6と配列番号 1 6はチヤィロコメノゴミムシダマ シ由来不凍蛋白質およぴ INP96の配列を示すが、 どちらの蛋白質においても - Thr- Xxx-Thr-の配列が 4回繰り返されていることがわかる。 また、 近年の研究から氷 結晶と不凍蛋白質の結合において、 不凍蛋白質と氷結晶面上の酸素原子との間の 水素結合よりは、 むしろ氷結晶面と不凍蛋白質表面の形状の相補性の方が優性で あることが示されており、 不凍蛋白質の氷結晶結合面に存在する水素結合性残基 を Valに置換しても不凍活性はほとんど変化しないことが示されている （Haymet ADJ et al.， 1998, FEBS Lett. , 430： 301 - 306 ; Chao H et al. , 1997, Biochemistry 36： 14652-14660) 。 それゆえ、 - Thr- Xxx- Thr-の配列中の Thrを Valに置換しても -Thr- Xxx- Thr-の場合と同様の活性を示す。 また、 Spruce Budwormにおいては配列中に含まれる -Thr- Xxx-Thr- (Thrを Valに置換されている ものも含む） の回数が 4〜6回のァイソマーが数種類見つかつており （Doucet D et al. , 2000， Eur. J. Biochem. , 267： 6082 - 6088 ; Leinala EK et al. , 2002, J. Biol. Chem. , 277： 33349-33352) 、 それらがいずれも不凍活性を有している ことが示されており、 - Thr - Xxx_Thr- (Thrを Valに置換されているものも含む） の回数が 4回に限定されたものではなく、 複数回繰り返されていれば不凍活性を 有することが示されている。 また、 -Thr - Xxx- Thr - （Thrを Valに置換されている ものも含む） の回数を増やすことで不凍活性が強くなることが示されており (Doucet D et al. , 2000, Eur. J. Biochem.， 267： 6082 - 6088 ; Leinala EK et al. , 2002, J. Biol. Chem. ' 277： 33349-33352) 、 配列番号 1 3で表されるァ ミノ酸配列の繰り返しの回数を変えることで、 任意の強さの不凍活性を有する不 凍蛋白質の作成が可能である。 FIG. 5 and SEQ ID NO: 15 and FIG. 6 and SEQ ID NO: 16 show the sequences of the antifreeze protein derived from Pseudococcidae and the INP96, and in both proteins, -Thr-Xxx-Thr- It can be seen that the sequence is repeated four times. Also, recent studies have shown that in the connection between ice crystals and antifreeze protein, the complementarity of the shape of the ice crystal surface and the surface of the antifreeze protein rather than the hydrogen bond between the antifreeze protein and oxygen atoms on the ice crystal surface. Has been shown to be more dominant, and it has been shown that the antifreeze activity hardly changes even if the hydrogen bonding residue present on the ice crystal binding surface of the antifreeze protein is replaced with Val ( Haymet ADJ et al., 1998, FEBS Lett., 430: 301-306; Chao H et al., 1997, Biochemistry 36: 14652-14660). Therefore, even when Thr in the sequence of -Thr-Xxx-Thr- is replaced with Val, the same activity as that of -Thr-Xxx-Thr- is exhibited. In Spruce Budworm, several isomers containing 4 to 6 times of -Thr-Xxx-Thr- (including those in which Thr is replaced by Val) are found in the sequence (Doucet D et al., 2000, Eur. J. Biochem., 267: 6082-6088; Leinala EK et al., 2002, J. Biol. Chem., 277: 33349-33352), all of which have antifreeze activity. -Thr-Xxx_Thr- (including those in which Thr is replaced by Val) is not limited to 4 times, but is antifreeze if repeated multiple times It is shown to have. It has also been shown that increasing the number of -Thr-Xxx-Thr-(including those in which Thr is replaced by Val) increases antifreeze activity (Doucet D et al., 2000, Eur J. Biochem., 267: 6082-6088; Leinala EK et al., 2002, J. Biol. Chem. '277: 33349-33352), the number of repetitions of the amino acid sequence represented by SEQ ID NO: 13. By changing the temperature, it is possible to create an antifreeze protein having an antifreeze activity of any strength.
これら蛋白質またはぺプチドを製造するには、 常法の遗伝子組み換え技術を用 いることができる。 すなわち、 これら蛋白質またはぺプチドをコ一ドする DNAを 調製し、 該 DNAを発現ベクターに導入し、 このベクターを用いて大腸菌等の適当 な宿主を形質転換し、 この形質転換体を培養することにより上記不凍機能を有す る蛋白質またはぺプチドを得ることができる。  To produce these proteins or peptides, conventional gene recombination techniques can be used. That is, a DNA encoding these proteins or peptides is prepared, the DNA is introduced into an expression vector, an appropriate host such as Escherichia coli is transformed with the vector, and the transformant is cultured. Thus, a protein or peptide having the above-mentioned antifreeze function can be obtained.
また、 他の製造方法としては、 ぺプチド合成機を用いて本発明の蛋白質または ぺプチドを合成してもよいし、 水核蛋白質またはその C末端ドメインぺプチダー ゼまたは酸、 アル力リ等の加水分解試薬を用いて限定的に部分分解することによ つても製造しうる。 なお、 この部分分解を行う場合においては、 不凍機能を有す る蛋白質のほかに不凍機能を有しないぺプチドも同時に生成しうるが、 少なくと も一定長以上の長さを有し、 配列番号 1 2で表されるアミノ酸配列を有する蛋白 質は不凍機能を有する蛋白質と見なしうる。 該蛋白質を常法により精製してもよ いが、 これ以外の蛋白質またはペプチドを含有していてもよく、 特に精製しなく とも不凍機能を発揮しうる。  Further, as another production method, the protein or peptide of the present invention may be synthesized using a peptide synthesizer, or a water core protein or its C-terminal domain peptide or an acid, It can also be produced by limited partial decomposition using a hydrolysis reagent. In the case of performing this partial decomposition, a peptide having no antifreeze function may be produced at the same time as a protein having an antifreeze function, but it has a length of at least a certain length. The protein having the amino acid sequence represented by SEQ ID NO: 12 can be regarded as a protein having an antifreeze function. The protein may be purified by a conventional method, but may contain other proteins or peptides, and can exhibit an antifreeze function without purification.
この際、 得られる蛋白質等が不凍機能を有するか否かは、 上記したように、 こ れら蛋白質等の水溶液において、 凍結温度で上記パイビラミッド型氷結晶の有無 を観察することにより、 簡単迅速にスク リーニングすることができる。 INP96の 不凍活性は、 再結晶阻害活性を顕微鏡で観察すること、 または、 凝固点降下度を 水点降下法浸透圧計で測定することにより確認できる。 At this time, whether or not the obtained protein or the like has an antifreeze function is determined, as described above, by the presence or absence of the aforementioned pyramid-shaped ice crystals at the freezing temperature in an aqueous solution of the protein or the like. By observing, the screen can be screened easily and quickly. The antifreeze activity of INP96 can be confirmed by observing the recrystallization inhibitory activity with a microscope or by measuring the degree of freezing point depression with a water point depression osmometer.
また、 本発明の不凍蛋白質またはペプチドは、 前述したように、 氷の再結晶化 防止剤ないしは凝固点降下剤として使用できる。 具体的な用途としては、 例えば アイスクリームまたは冷凍食品などに混入することによりその品質を持続させる ために使用できる。 さらに、 近年、 エネルギー密度が大きい氷スラリーを熱媒体 として使用する冷熱供給システムまたは冷熱蓄熱等が提案されているが、 これら においては、 氷の再結晶による配管系の閉塞の問題があり、 本発明の不凍蛋白質 は氷の再結晶化を有効に防ぐものであるから、 この問題を解決するために有望な 手段となり うる。 このほか卵子や精子などの細胞の低温長期保存も応用技術とし て期待できる。  Further, as described above, the antifreeze protein or peptide of the present invention can be used as an ice recrystallization inhibitor or a freezing point depressant. As a specific application, for example, it can be used to maintain its quality by being mixed into ice cream or frozen food. Furthermore, in recent years, a cold heat supply system or a cold heat storage system using an ice slurry having a large energy density as a heat medium has been proposed. Since antifreeze proteins effectively prevent recrystallization of ice, they can be a promising tool to solve this problem. In addition, long-term storage of cells such as eggs and sperm at low temperatures can be expected as an applied technology.
また、 本発明の不凍蛋白質遺伝子の応用としては植物および魚類などへの不凍 蛋白質遺伝子の組込みにより、 それらの耐冷性を向上させることが期待される。 図面の簡単な説明  In addition, as an application of the antifreeze protein gene of the present invention, it is expected that by incorporating the antifreeze protein gene into plants, fishes and the like, their cold resistance will be improved. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 バイビラミツド型氷結晶の模式図である。  Figure 1 is a schematic diagram of a viviramid ice crystal.
図 2は、 チヤィ口コメノゴミムシダマシ由来不凍蛋白質の立体構造モデルを示 した図である。  FIG. 2 is a diagram showing a three-dimensional structural model of an antifreeze protein derived from the locust beetle, Chinoen beetle.
図 3は、 チヤィ口コメノゴミムシダマシ由来不凍蛋白質の氷結晶面結合モデル を示した図である。  FIG. 3 is a diagram showing an ice crystal surface binding model of an antifreeze protein derived from the cynomolgus duck beetle.
図 4は、 氷核蛋白質 inaZの N末端領域、 中心領域おょぴ C末端領域の各ァミノ 酸配列、 ならびに該中心領域中の INP96部分 (下線部) を示す図である。  FIG. 4 is a view showing the N-terminal region of the ice core protein inaZ, the amino acid sequences of the central region and the C-terminal region, and the INP96 portion (underlined portion) in the central region.
図 5は、 INF96のアミノ酸配列を示す図である。 下線部は TXT配列の部分を示す。 図 6は、 チヤイロコメノゴミムシダマシ由来不凍蛋白質のアミノ酸配列を示す 図である。 下線部は TXT配列の部分を示す。  FIG. 5 shows the amino acid sequence of INF96. The underlined part indicates the part of the TXT array. FIG. 6 is a diagram showing the amino acid sequence of an antifreeze protein derived from the locust beetle, Dermatophagoides farinae. The underlined part indicates the part of the TXT array.
図 7は、 INP96の DNA配列を示す図である。  FIG. 7 shows the DNA sequence of INP96.
図 8は、 キチンカラムにおける INP96の溶出パターンを示すクロマトグラムで ある。  FIG. 8 is a chromatogram showing the elution pattern of INP96 on a chitin column.
図 9は、 キチンカラムにおける INP96の溶出フラクションの SDS- PAGEである。 図 1 0は、 INP96の 9mg/ml水溶液について観測されたパイビラミッド型氷結晶 の写真である。 FIG. 9 is an SDS-PAGE of the elution fraction of INP96 on a chitin column. Figure 10 is a photograph of a piviramid-type ice crystal observed for a 9 mg / ml aqueous solution of INP96.
図 1 1は、 ニヮトリ卵白リゾチームと INP96についての凝固点の濃度依存性の の測定結果を示す図である。  FIG. 11 is a graph showing the results of measuring the concentration dependence of the freezing point of chicken egg white lysozyme and INP96.
本明細書は、 本願の優先権の基礎である特願 2 0 0 3 - 0 3 8 7 5 1号の明細書に記載 された内容を包含する。 発明を実施するための最良の形態 This description includes part or all of the contents as disclosed in the description of Japanese Patent Application No. 2003-038751, which is a priority document of the present application. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施例を示すが、 本発明は特にこれにより限定されるものでは ない。 実施例 1 不凍活性の確認  Hereinafter, examples of the present invention will be described, but the present invention is not particularly limited thereto. Example 1 Confirmation of antifreeze activity
( 1 ) 検体試料 (不凍蛋白質 INP96の調製)  (1) Sample (preparation of antifreeze protein INP96)
( a ) プラスミ ド pTYB12INP96の調製  (a) Preparation of plasmid pTYB12INP96
まず INP96をコードする DNAをデザインした。 氷核蛋白質の繰り返し配列中に現 れる 96残基のァミノ酸配列をそれに対応する塩基配列に変換した。 この際、 大腸 菌の繁用コドンを参考にした (Bennetzen, JL, Hall, BD, Codon selection in yeast, J. Biol. Chem. , 257, 3026 - 3031 (1982) ) 。 このようにデザインした INP96のアミノ酸配列とそれをコードする DNAの塩基配列を図 7、 配列番号 1 7お よび配列番号 5に示す。 この DNAを全合成することは困難であったため、 互いに 一部相補的な 4本の DNAに分割し、 DNA自動合成機によって INP96aalf (配列番号 6 ) 、 INP96aalr (配列番号 7 ) 、 INP96aa2f (配列番号 8 ) および INP96aa2r (配列番号 9 ) を合成した。  First, we designed the DNA encoding INP96. The amino acid sequence of 96 residues appearing in the repetitive sequence of the ice core protein was converted to the corresponding nucleotide sequence. At this time, common codons of E. coli were referred to (Bennetzen, JL, Hall, BD, Codon selection in yeast, J. Biol. Chem., 257, 3026-3031 (1982)). The amino acid sequence of INP96 thus designed and the nucleotide sequence of the DNA encoding it are shown in FIG. 7, SEQ ID NO: 17 and SEQ ID NO: 5. Since it was difficult to completely synthesize this DNA, it was divided into four DNAs partially complementary to each other, and INP96aalf (SEQ ID NO: 6), INP96aalr (SEQ ID NO: 7), INP96aa2f (SEQ ID NO: 8) and INP96aa2r (SEQ ID NO: 9) were synthesized.
次いで、 これら 4本の合成 DNAをその相補的領域でァニールさせると共に、 結 合させるために PCR反応を行った。 この反応は、 INP96aalfと INP96aa2r 600nM、 INP96aalrと INP96aa2f 1ηΜ、 dNTP (4種のデォキシヌクレオチド三リン酸の混合 溶液） 200 mM、 ェクスパンド■ハイ · ヒィデリティ PCRシステム （Expand High Fidel ity PCR system) (Roche) に添付されたバッファー ( IX) 、 ェクスパン ド · HiFi DNAポリメラーゼ （Expand HiFi DNA polymerase) 1. 3Uを含む反応溶 液 50mlで行った。 この反応溶液を 0. 2mlの PCR反応用チューブに 6本作り、 タカラ PCR サーマル サイクラ一 MP (Takara PCR Thermal cycler MP) を用いて、 第 一ステップ ： 94°Cで 2分、 第二ステップ ： 94°Cで 30秒 （変性） 、 スロープを設定 して 1分かけて 65°Cまで温度を下げ、 65°Cで 30秒 （アニーリング） 、 72°Cで 1. 5 分 （伸長） のサイクルを 35回、 第三ステップ： 72°Cで 5分で PCR反応を行った。 次いで互いに連結させることによって作製した二本鎖 DNAを pT7Blue T-べクタ ― (Novagen社） と連結し、 該ベクターを用いて大腸菌 DH5a ( (独）産業技術総合研 究所 ·生物機能工学研究部門で保存） を形質転換した。 1 mM IPTG、 0. 02% X- Gal、 lOOmg/mlアンピシリンを含む LBプレートに形質転換体を塗布し、 37°Cでおよそ 20 時間培養した。 出現した白色のコロニーの内 8コロニーを lOOmg/mlアンピシリン を含む LB培地 3mlで 37°Cでー晚試験管で培養した後、 クォンタムプレブ · プラス ミ ド · ミニプレブ ' キッ ト ( QuantumPrep Plasmid MiniPrep kit ) (Bio-Rad 社） を用いてプラスミ ドを調製し、 制限酵素による切断パターンから目的の DNA を含んでいると思われるプラスミ ドを保有した形質転換体 3個を判別した。 これ らについて、 ビッグダイ ' ターミネータ一 *サイクノレ ' シークェンシング ' レデ Λ ' ~ · リアクショ ン - キッ ト ( BigDye Terminator Cycle Sequencing Ready Reaction kit) ver. 2. 0 (Applied Biosystems社） を用いて塩基配列を確認した。 その結果、 合成一本鎖 DNA中の合成ミスまたは PCR中の誤りによると考えられる塩 基置換が認められたので、 これを 2回目の PCRによって修正した。 Next, these four synthetic DNAs were annealed in their complementary regions, and a PCR reaction was performed to ligate them. This reaction was performed using INP96aalf and INP96aa2r 600 nM, INP96aalr and INP96aa2f 1ηΜ, dNTP (mixed solution of four types of deoxynucleotide triphosphates) 200 mM, and Expand High Fidelity PCR system (Roche ), A reaction solution containing 1.3 U of Expand HiFi DNA polymerase (Expand HiFi DNA polymerase). Performed with 50 ml of liquid. Prepare 6 tubes of this reaction solution in a 0.2 ml PCR reaction tube and use Takara PCR Thermal cycler MP (Takara PCR Thermal cycler MP). First step: 2 minutes at 94 ° C, Second step: 9 Cycle at 4 ° C for 30 seconds (denaturation), ramp down to 65 ° C over 1 minute, cycle at 65 ° C for 30 seconds (annealing), 72 ° C for 1.5 minutes (extension) 35 times, Third step: PCR reaction was performed at 72 ° C for 5 minutes. Subsequently, the double-stranded DNAs produced by ligation were ligated to pT7Blue T-vector (Novagen), and Escherichia coli DH5a (Biotechnology Engineering Research Division, National Institute of Advanced Industrial Science and Technology) ) Was transformed. The transformant was spread on an LB plate containing 1 mM IPTG, 0.02% X-Gal, 100 mg / ml ampicillin, and cultured at 37 ° C for about 20 hours. Eight of the white colonies that emerged were cultured in 3 ml of LB medium containing lOOmg / ml ampicillin in a test tube at 37 ° C at 37 ° C. ) (Bio-Rad) was used to prepare plasmids, and three transformants carrying plasmids that were thought to contain the target DNA were identified based on the restriction enzyme cleavage pattern. For these, the base sequence was determined using Big Dye Terminator Cycle Sequencing レ Sequencing レ Ready ~ ~ Reaction-Kit (BigDye Terminator Cycle Sequencing Ready Reaction kit) ver. 2.0 (Applied Biosystems). confirmed. As a result, a base substitution that was considered to be due to a synthesis error in the synthesized single-stranded DNA or an error in the PCR was recognized. This was corrected by the second PCR.
2回目の PCR反応に利用したプライマーの配列は INP96f2 (配列番号 1 0 ) およ び INP961-2 (配列番号 1 1 ) である。  The sequences of the primers used in the second PCR reaction were INP96f2 (SEQ ID NO: 10) and INP961-2 (SEQ ID NO: 11).
この PCR反応では、 PCR反応中にエラーが起こる可能性をできるだけ低くするた めにエラー頻度が低いことが知られている K0D ポリメラーゼ (KOD polymerase) (東洋紡社） を用いた。 PCRは 5000倍に希釈した上記のプラスミ ド溶液、 dNTP 200mM、 塩化マグネシウム 1 mM、 INP96f2、 INP96r2それぞれ 300nM、 K0D-プラス 一 (KOD -Plus-) に添付されたバッファー （IX) 、 K0D-プラス-ポリメラーゼ (KOD -Plus- polymerase) 1 Uを含む反応溶液 50mlで行った。 この反応溶液を 0. 2mlの PCR反応用チューブに 2本作り、 Takara PCR Thermal cycler MPを用いて、 第一ステップ： 94°Cで 2分、 第二ステップ： 94°Cで 15秒、 60。Cで 30秒、 68°Cで 1分 (伸長） のサイクルを 30回、 第三ステップ： 68°Cで 5分で PCR反応を行った。 PCR反応液を用いてヌシーブ GTG ァガロース （NuS ieve GTG Agarose) (タカラ 社） を用いたァガロース電気泳動を行い、 およそ 300 bpの位置に出現した DNAの パンドをァガロースゲルから回収した。 次いで、 回収した DNAを、 あらかじめ Eco RVで切断した pZEr02ベクター （Invtrogen社） と連結し、 該ベクターを用いて大 腸菌 DH5aを形質転換した。 1 mM IPTG、 50mg/mlカナマイシンを含む LS- LB ( 1% ト リプトン （Trypton) 、 0. 5%ィース ト 'エキス トラク ト （Yeast Extract) 、 0. 5% 塩化ナトリウム） プレートに形質転換体を塗布し、 37°Cでおよそ 20時間培養した, 出現したコロニーの内 8コロニーを 50mg/mlカナマイシンを含む LS- LB培地 3mlで 37°Cで一晩試験管で培養した後、 クォンタムプレブ · プラスミ ド · ミニプレブ · キット （QuantumPrep Plasraid MiniPrep kit) を用いてプラスミ ドを調製し、 制 限酵素による切断パターンから目的の DNAを含んでいると思われるプラスミ ドを 保有した形質転換体 3個を判別した。 これらについて、 ビッグダイ · ターミネ一 タ一 · サイクル · シークェンシング · レディ一 · リアクション ' キッ ト ver. 2. 0 を用いて塩基配列を確認した。 その結果、 図 7に示した目的の塩基配列を有する DNAが揷入されたプラスミ ド (PZEr02INP96) を得ることができた。 次に該プラス ミ ドを保持する形質転換体を 50mlの培養液で培養し、 クォンタムプレブ · プラス ミ ド · ミエプレブ · キット (Bio-Rad社) を用いてプラスミ ドを調製した。 In this PCR reaction, KOD polymerase (KOD polymerase) (Toyobo Co., Ltd.), which is known to have a low error frequency, was used in order to minimize the possibility of errors occurring during the PCR reaction. The PCR was performed using the above plasmid solution diluted 5000 times, dNTP 200 mM, magnesium chloride 1 mM, INP96f2, INP96r2 300 nM each, buffer (IX) and K0D-plus- attached to K0D-Plus- (KOD-Plus-). The reaction was performed with 50 ml of a reaction solution containing 1 U of a polymerase (KOD-Plus-polymerase). Make two of this reaction solution in a 0.2 ml PCR reaction tube, and use Takara PCR Thermal cycler MP for the first step: 2 minutes at 94 ° C, and the second step: 15 seconds at 94 ° C, 60. The PCR reaction was performed 30 times at 30 ° C. and 1 minute (extension) at 68 ° C. Third step: The PCR reaction was performed at 68 ° C. for 5 minutes. Using a PCR reaction solution, agarose electrophoresis was performed using NuSieve GTG Agarose (Takara), and a DNA band that appeared at a position of about 300 bp was recovered from the agarose gel. Next, the recovered DNA was ligated to a pZEr02 vector (Invtrogen) cut in advance with Eco RV, and Escherichia coli DH5a was transformed using the vector. Transformants were plated on LS-LB (1% Trypton, 0.5% Yeast Extract, 0.5% sodium chloride) plates containing 1 mM IPTG and 50 mg / ml kanamycin. After culturing at 37 ° C for about 20 hours, 8 of the emerged colonies were cultured in 3 ml of LS-LB medium containing 50 mg / ml kanamycin at 37 ° C overnight in a test tube. Plasmids are prepared using the Plasmid MiniPreb Kit (QuantumPrep Plasraid MiniPrep kit), and three transformants carrying the plasmid that is thought to contain the target DNA are identified based on the restriction enzyme cleavage pattern. did. For these, the base sequences were confirmed using Big Dye, Terminator, Cycle, Sequencing, Ready, and Reaction 'kit ver. 2.0. As a result, it was possible to obtain the DNA plasmid that is揷入(P ZEr02INP96) having the nucleotide sequence of interest shown in FIG. Next, the transformant holding the plasmid was cultured in a 50-ml culture solution, and a plasmid was prepared using a Quantumpreb-plasmid-mieprep kit (Bio-Rad).
次に、 得られた DNAを用いて INP96を大腸菌で発現させるために、 IMPACT - CN シ ステム (system IMPACT - CN system) (New England Biolabs社) を用いた。 該シ ステムでは、 目的の蛋白質をキチン結合蛋白質、 プロテアーゼであるインティン との融合蛋白質として生産する。 このため、 該システムで用いられる発現べクタ 一にはあらかじめ、 発現蛋白質の精製を容易にするキチン結合ドメインをコード する塩基配列、 および目的の蛋白質を切り離すための自己消化プロテアーゼ · ィ ンティンをコードする塩基配列が組み込まれてある。 pZEr02INP96を Nde Iと Eco RIで切断し、 およそ 300 bpの INP96をコードする DNAをヌシーブ GTG ァガロースを 用いたァガロース電気泳動により分離 '回収した。 一方、 該システムに含まれて いる発現ベクター PTYB12を Nde Iと Eco RIで切断した。 両者をタカラ · DNA · リゲ イシヨン 'キット （Takara DNA Ligat ion kit) (タカラ社） を用いて、 添付の プロ トコルに従って連結し、 大腸菌 DH5aを形質転換した。 100mg/mlアンピシリン を含む LBプレートに形質転換体を塗布し、 37°Cでおよそ 18時間培養した。 出現し たコロニーの内 18コロニーを lOOmg/mlアンピシリンを含む LB培地 3mlで 37°Cで一 晚試験管で培養した後、 クォンタムプレブ · プラスミ ド · ミニプレブ · キッ トを 用いてプラスミ ドを調製し、 制限酵素による切断パターンから目的の DNAを含ん でいると思われるプラスミ ドを保有した形質転換体 10個を判別した。 これらのう ち 1つの形質転換体を 50mlの培養液で培養し、 クォンタムプレブ 'プラスミ ド . ミニプレブ ·キットを用いてプラスミ ドを調製した。 さらに、 ビッグダイ · ター ミネ一ター ·サイクル ' シークェンシンク' · レディ一 · リアクション · キッ ト ver. 2. 0を用いて塩基配列を確認した。 その結果、 図 7に示した目的の塩基配列 を有する DNAが pTYB12に挿入されたプラスミ ド （pTYB12INP96) を得ることができ た。 Next, in order to express INP96 in E. coli using the obtained DNA, IMPACT-CN system (New England Biolabs) was used. In this system, a target protein is produced as a fusion protein with a chitin-binding protein and a protease, intin. For this reason, the expression vector used in the system encodes, in advance, a nucleotide sequence encoding a chitin-binding domain that facilitates purification of the expressed protein, and an autolyzing protease-tin for cutting off the target protein. The base sequence has been incorporated. pZEr02INP96 was digested with NdeI and EcoRI, and the DNA encoding about 300 bp of INP96 was separated and recovered by agarose electrophoresis using nucleic acid GTG agarose. On the other hand, the expression vector PTYB12 contained in the system was cut with NdeI and EcoRI. Both were ligated using a Takara DNA Ligation kit (Takara) according to the attached protocol to transform Escherichia coli DH5a. The transformant was spread on an LB plate containing 100 mg / ml ampicillin, and cultured at 37 ° C for approximately 18 hours. Appear 18 colonies were cultured in 3 ml of LB medium containing lOOmg / ml ampicillin at 37 ° C in a test tube, and then prepared with Quantum Preb, Plasmid, Mini Preb Kit and prepared. From the pattern of cleavage by the restriction enzyme, 10 transformants carrying the plasmid likely to contain the target DNA were identified. One of these transformants was cultured in a 50 ml culture solution, and a plasmid was prepared using Quantumpreb 'Plasmid. Minipreb kit. In addition, the base sequence was confirmed using Big Dye Terminator Cycle 'Sequen Sync', Ready, Reaction, Kit ver. 2.0. As a result, a plasmid (pTYB12INP96) in which the DNA having the target nucleotide sequence shown in FIG. 7 was inserted into pTYB12 was obtained.
( b ) 大腸菌による INP96の発現  (b) Expression of INP96 by E. coli
INP96はキチン結合ドメインおよぴィンティンとの融合蛋白質として大腸菌体 内に発現させた。 プラスミ ド pTYB12INP96で大腸菌 ER2566 (DE3) (New England Biolab社) を形質転換した。 プラスミ ド pYT12INP96にはアンピシリン耐性遺伝子 が導入されているため、 アンピシリン含有 LB寒天培地に大腸菌を広げ、 ー晚 37°C でィンキュベートすることで形質転換体を選択した。 形成されたコロニーの一つ を 100 ,u g/mlのアンピシリンを含んだ LB培地 15 mlに植え 28°Cでー晚培養した。 この培養液を 100 g/mlのアンピシリンぉよび 0. 5 mMィソプロピル - D (—）- チォガラク トビラノシドを含んだ ΤΒ培地 1. 5 1に植え継ぎし、 さらに 28°Cでー晚 培養した。 培養液を 3600 X g、 4°Cで 15分遠心分離し、 菌体を回収した。 菌体を 20 mM トリス -塩酸緩衝液 I ImM エチレンジァミン四酢酸ニナトリ ウム (PH 8. 0)に懸濁し、 氷中で超音波破碎した。 これを 11900 X g、 4°Cで 60分遠心分離 し、 可溶性画分と不溶性画分に分離した。 INP96 was expressed in Escherichia coli as a fusion protein with chitin-binding domain and tintin. Escherichia coli ER2566 (DE3) (New England Biolab) was transformed with plasmid pTYB12INP96. Since an ampicillin resistance gene was introduced into plasmid pYT12INP96, transformants were selected by spreading Escherichia coli on LB agar medium containing ampicillin and incubating at -37 ° C. One of the formed colonies was inoculated in 15 ml of LB medium containing 100 ug / ml of ampicillin, and cultured at 28 ° C. This culture was subcultured into a medium 1.51 containing 100 g / ml of ampicillin and 0.5 mM isopropyl-D (-)-thiogalactobyranoside, and further cultured at 28 ° C. The culture was centrifuged at 3600 X g at 4 ° C for 15 minutes to collect the cells. The cells 20 mM Tris - was suspended in HCl buffer I ImM Echirenjiamin tetraacetate Ninatori um (P H 8. 0), and ultrasonic Yabu碎in ice. This was centrifuged at 11,900 X g at 4 ° C for 60 minutes to separate a soluble fraction and an insoluble fraction.
( c ) INP96の精製 ' (c) Purification of INP96 ''
可溶性画分から抽出したキチン結合ドメインおよびィンティンとの融合蛋白質 の状態の INP96は 20mM トリス-塩酸緩衝液 I ImM エチレンジァミン四酢酸ニナト リウム / 0. 5M NaCl水溶液（pH8. 0)で緩衝化したキチンカラムに吸着させた。 力 ラムボリュームの 20倍量の 20mM トリス-塩酸緩衝液 I ImM エチレンジァミン四 酢酸-ナトリウム / 0. 5M NaCl水溶液（ρΗ8· 0)を流し、 その後、 カラムボリユー ムの 3倍量の 20mM トリス-塩酸緩衝液 I ImM エチレンジァミン四酢酸ニナトリウ ム I 0. 5M NaCl / 50mM -メルカプトエタノール水溶液（pH8. 0)を流し、 室温で 48時間以上放置し、 INP96と、 キチン結合ドメインおよびインティンからなるタ グの部分とを切断した。 その後、 3倍量の 20mM トリス-塩酸緩衝液 I ImM ェチレ ンジァミン四酢酸ニナトリウム / 0. 5M NaCl水溶液（ρΗ8· 0)を流し INP96を溶出さ せた。 この際、 タグが切断された ΙΝΡ96のみが溶出してくる。 図 8にクロマトグ ラムを示した。 各フラクッションのポリユームは約 1. 5〜2 mlである。 INP96 in the form of a fusion protein with chitin-binding domain and intin extracted from the soluble fraction was placed on a chitin column buffered with 20 mM Tris-HCl buffer I ImM ninatrium ethylenediaminetetraacetate / 0.5 M NaCl aqueous solution (pH 8.0). Adsorbed. 20 mM Tris-HCl buffer I 20 times the volume of the column I ImM Sodium ethylenediaminetetraacetate-sodium / 0.5 M NaCl aqueous solution (ρΗ80), then 20 mM Tris-HCl buffer 3 times the column volume I ImM Ethylenediamine tetraacetate sodium Then, an aqueous solution of 0.5 M NaCl / 50 mM-mercaptoethanol (pH 8.0) was allowed to flow, and the mixture was allowed to stand at room temperature for 48 hours or more to cut INP96 and a tag portion comprising a chitin binding domain and intin. Thereafter, a three-fold volume of 20 mM Tris-HCl buffer I ImM ethylenediamine tetrasodium disodium / 0.5 M NaCl aqueous solution (ρΗ80) was flowed to elute INP96. At this time, only # 96 with the cleaved tag elutes. Figure 8 shows the chromatogram. Each hula cushion has a polyyume of about 1.5 to 2 ml.
( d ) ドデシル硫酸ナトリウム一ポリアクリルアミ ドゲル電気泳動  (d) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
一般的な手順に従い、 電気泳動装置 （ATT0) 用いて精製した INP96を 15% ポリ アクリルアミ ドゲルで泳動した。 分子量測定のためブロード ' レンジ SDS- PAGE スタンダード (broad range SDS - PAGE standard ) (BIO - RAD) を同時に泳動した ( サンプル緩衝液は、 0. 065 M トリス-塩酸緩衝液 （pH6. 8) / 2 % ドデシル硫酸ナ トリ ウム I 10 % ショ糖 / 5 % β -メルカプトェタノール I 0. 001 % ブロモフ ェノールブルーを用いた。 濃縮ゲルは 0. 5 Μ トリス-塩酸緩衝液 (ρΗ 6. 8) 0. 75 ml、 30 % ァク リルアミ ド I ビス (37. 5 ： 1) 混合液 0. 45 ml、 10 % ドデシノレ 硫酸ナトリゥム 0. 12 ml、 蒸留水 1. 78 mlを混合して作製し、 分離ゲルは 1. 5 M トリス-塩酸緩衝液 (pH 8. 8) 2. 25 ml、 30% ァク リルアミ ド I ビス (37. 5 ： 1) 混合液 4, 5 ml、 10 % ドデシル硫酸ナトリウム 0. 36 ml、 蒸留水 1. 89 mlを混合 して作製した。 泳動用緩衝液は、 ト リ ス (ヒ ドロキシメチル) ァミノメタン 3. 03 g、 グリシン 14. 4 gおよぴドデシル硫酸ナトリ ウム l gを合計 1 1の蒸留水 中に溶解させたものを用いた。 ゲルの染色はメタノール 20 ml、 硫酸アンモニゥ ム 12. 5g、 リン酸 2. 5ml、 0. 04 % クマシーブリ リアントブルー G - 250を 100ml の蒸留水の溶液に溶かした染色液を用いて一晩行い、 蒸留水で脱染色した。 図 9 より、 分子量 11 KDa の INP96が純度高く精製されているのが確認できる。  According to the general procedure, the purified INP96 was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (ATT0). Broad range SDS-PAGE standard (BIO-RAD) was run simultaneously for molecular weight measurement (sample buffer was 0.065 M Tris-HCl buffer (pH 6.8) / 2 % Sodium dodecylsulfate I 10% sucrose / 5% β-mercaptoethanol I 0.001% bromophenol blue concentrated gel 0.5 0 Tris-HCl buffer (ρΗ 6.8) 0 75 ml, 0.45 ml of 30% acrylamide Ibis (37.5: 1) mixture, 0.12 ml of 10% sodium dodecinole sulfate and 1.78 ml of distilled water are mixed and prepared. The gel is 1.5 M Tris-HCl buffer (pH 8.8) 2.25 ml, 30% acrylamide Ibis (37.5: 1) mixture 4, 5 ml, 10% sodium dodecyl sulfate 0 The mixture was prepared by mixing 36 ml of distilled water and 1.89 ml of distilled water The electrophoresis buffer was 3.03 g of tris (hydroxymethyl) amino methane, 14.4 g of glycine, and the like. A solution of sodium dodecyl sulfate lg in a total of 11 distilled water was used for gel staining: methanol 20 ml, ammonium sulfate 12.5 g, phosphoric acid 2.5 ml, 0.04% Coomassie brilliant Performed overnight using a staining solution prepared by dissolving Blue G-250 in 100 ml of distilled water, and destained with distilled water From Fig. 9, it can be confirmed that INP96 having a molecular weight of 11 KDa was highly purified. .
( 2 ) バイピラミダル氷結晶の観察  (2) Observation of bipyramidal ice crystals
( a ) INP96を 20mM トリス-塩酸緩衝液 I ImM エチレンジァミン四酢酸ニナトリ ゥム I 0. 5M NaCl水溶液（pH8. 0)に対して透析し、 緩衝液置換を行った後に 9mg/mlになるまで限外濾過により濃縮を行った。 なお、 注目する検体液が不凍活 性を有するか否かの評価は、 わずか lulの液に対しても低温顕微鏡下でのバイピ ラミツド型氷結晶観察実験をすることで行い得る。  (a) Dialyze INP96 against 20 mM Tris-HCl buffer I ImM ethylenediaminetetraacetate sodium acetate I 0.5 M NaCl aqueous solution (pH 8.0), replace buffer, and limit to 9 mg / ml. Concentration was performed by external filtration. The evaluation of whether or not the sample solution of interest has antifreeze activity can be performed by conducting an experiment on the observation of bipiramid-type ice crystals under a low-temperature microscope even for a small amount of the solution.
( b ) 上記のように調製した試料溶液 lulをライカ社製 DMLB100型顕微鏡（Leica DMLB 100 photomicroscope)の直径 16mmのカバーガラス上に滴下した。 これをそ のままもう 1枚の直径 12. 5mmのカバーガラスによりはさみ、 これを DMLB100型顕 微鏡のステージ部に設置した冷却箱内にセットした。 冷却箱の上下には直径 lmm の光取り入れ穴をあけ、 顕微鏡光源からの光は下側の穴から箱内を通り上側の穴 を抜けてレンズに入光させるようにした。 この上下の穴により規定される光軸上 に検体液をセットすることで、 光軸上にある検体液中の物質を顕微鏡観察するこ とができる。 検体液がセットされた冷却箱の中の温度は、 リンカム社製 LK600温 度制御装置 (Linkam LK600 temperature controller) により +/— 0. 1°Cの誤差で 制御される。 室温下で検体液をセットした後、 温度制御装置により冷却箱内の温 度を毎秒 0. 2°Cで- 22°Cまで下降させた。 およそ- 14°Cから- 22。Cの間の温度のどこ かで検体液の全体が凍結する。 凍結の後に毎秒 0. 2°Cで冷却箱内温度を上昇させ 零度で上昇を停止し、 そのまま 1〜 10秒程度の間、 -3°Cを維持していると凍結が 溶け、 無数のきれつの入った氷結晶状態を経たのちに、 数えられる程度の氷結晶 が水中に浮かぶのが観測された。 その瞬間に、 冷却箱内の温度を- 2°C〜3°C程度 に下降させて止め、 氷結晶の形状を観察した。 観察結果を図 1 0に示す。 試験に 使用した INP96においてバイビラミッド型の氷晶が観察され、 この蛋白質は不凍 活性を有することが確認された。 (b) A sample solution lul prepared as described above was added to a Leica DMLB100 microscope (Leica The solution was dropped on a cover glass having a diameter of 16 mm of a DMLB 100 photomicroscope). This was sandwiched by another cover glass with a diameter of 12.5 mm, and this was set in a cooling box installed on the stage of a DMLB100 microscope. Light intake holes with a diameter of lmm were drilled above and below the cooling box, and light from the microscope light source passed through the inside of the box from the lower hole, passed through the upper hole, and entered the lens. By setting the sample liquid on the optical axis defined by the upper and lower holes, the substance in the sample liquid on the optical axis can be observed with a microscope. The temperature in the cooling box in which the sample solution is set is controlled with an error of +/- 0.1 ° C by a Linkam LK600 temperature controller (Linkam LK600 temperature controller). After the sample solution was set at room temperature, the temperature in the cooling box was decreased from 0.2 ° C per second to -22 ° C by the temperature controller. Approximately -14 ° C to -22. Somewhere in the temperature between C, the whole sample freezes. After freezing, raise the temperature inside the cooling box at 0.2 ° C per second, stop rising at zero degree, and keep it at -3 ° C for about 1 to 10 seconds. After passing through a single ice crystal state, countable ice crystals were observed floating in the water. At that moment, the temperature inside the cooling box was lowered to about -2 ° C to 3 ° C and stopped, and the shape of the ice crystals was observed. The observation results are shown in FIG. In the INP96 used in the test, viviramid-shaped ice crystals were observed, confirming that this protein has antifreeze activity.
( c ) 20mM トリス-塩酸緩衝液 I ImM エチレンジァミン四酢酸ニナトリウム / 0. 5M NaCl水溶液（pH8. 0)に対して透析し、 緩衝液置換を行った後に 19 mg/mlにな るまで限外濾過により濃縮を行った。 様々な濃度に希釈した INP96のサンプル 50 i 1を用いて総浸透圧値を氷点降下法浸透圧計 (V0GEL) を用いて測定し、 凝固点 を算出した （図 1 1 ) 。  (c) 20 mM Tris-HCl buffer I ImM Dialyze against disodium ethylenediaminetetraacetate / 0.5 M NaCl aqueous solution (pH 8.0), replace with buffer, and limit to 19 mg / ml. Concentration was performed by filtration. The freezing point was calculated by measuring the total osmotic pressure using a freezing point osmometer (V0GEL) using INP96 samples 50 i1 diluted to various concentrations (Fig. 11).
図 1 1において対照化学物質であるニヮトリ卵白リゾチームは 0. 02°Cの範囲内 で直線的な凝固点降下を示しており、 これは一 的な化学物質のモル凝固点降下 現象に起因している。 一方、 INP96については 0. 12°Cまでの非直線的な凝固点降 下を示しており、 これは水溶液中の不凍蛋白質が特異的に氷結晶表面と相互作用 をする場合にのみ観測される。 この実験により、 INP96が不凍蛋白質様の凝固点 降下活性を示すことが確認された。  In Fig. 11, the control chemical, hen egg white lysozyme, shows a linear freezing point fall within the range of 0.02 ° C, which is due to the molar freezing point depression phenomenon of the chemical substance. On the other hand, INP96 shows a non-linear decrease in freezing point up to 0.12 ° C, which is observed only when the antifreeze protein in the aqueous solution specifically interacts with the ice crystal surface. . This experiment confirmed that INP96 exhibited antifreeze protein-like freezing point lowering activity.
本明細書中で引用した全ての刊行物、 特許及び特許出願をそのまま参考として 本明細書中に取り入れるものとする。 産業上の利用の可能性 All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial potential
本発明は、 氷核蛋白質の構造に基づいて、 不凍蛋白質を設計、 創出してそれら を供給するものである。 そして、 本発明においては不凍蛋白質または不凍べプチ ドを、 ペプチド合成法、 遺伝子工学または氷核蛋白質の部分分解等により生産す ることにより、 これらを容易にかつ大量に供給できる。 さらに、 季節による品質 のばらつきがなく、 一定の品質の不凍蛋白質または不凍べプチドを供給すること ができる。 したがって、 本発明は、 不凍蛋白質または不凍ペプチドの安定的な供 給、 ならびに不凍蛋白質またはぺプチドについての利用促進および応用研究の発 展に大いに寄与するものである。 配列表フリーテキスト  The present invention is to design, create and supply antifreeze proteins based on the structure of ice core proteins. In the present invention, antifreeze proteins or antifreeze peptides can be easily and massively supplied by producing them by peptide synthesis, genetic engineering, or partial decomposition of ice nucleus proteins. In addition, it is possible to supply antifreeze proteins or antifreeze peptides of consistent quality without seasonal quality variations. Therefore, the present invention greatly contributes to stable supply of antifreeze proteins or antifreeze peptides, promotion of utilization of antifreeze proteins or peptides, and development of applied research. Sequence listing free text
配列番号 5： INP96aaをコード DNA配列 SEQ ID NO: 5: DNA sequence encoding INP96aa
配列番号 6： INP96aalfをコード DNA配列を構築するためのプライマー SEQ ID NO: 6: primer for constructing DNA sequence encoding INP96aalf
配列番号 7： INP96aalrをコード DNA配列を構築するためのプライマー SEQ ID NO: 7: primer for constructing DNA sequence encoding INP96aalr
配列番号 8： INP96aa2fをコ一ド DNA配列を構築するためのプライマー SEQ ID NO: 8: Primer for constructing a DNA sequence encoding INP96aa2f
配列番号 9： INP96aa2rをコード DNA配列を構築するためのプライマー SEQ ID NO: 9: Primer for constructing DNA sequence encoding INP96aa2r
配列番号 10： INP96f2をコード DNA配列を構築するためのプライマー SEQ ID NO: 10: Primer for constructing DNA sequence encoding INP96f2
配列番号 11： INP96r2をコード DNA配列を構築するためのプライマー SEQ ID NO: 11: Primer for constructing DNA sequence encoding INP96r2
配列番号 12： Xaaは任意のァミノ酸残基を示す。 SEQ ID NO: 12: Xaa represents any amino acid residue
配列番号 13： Xaaは任意のアミノ酸残基を示す。 SEQ ID NO: 13: Xaa represents any amino acid residue
配列番号 15：合成ァミノ酸配列 SEQ ID NO: 15: Synthetic amino acid sequence
配列番号 16：合成ァミノ酸配列 SEQ ID NO: 16: Synthetic amino acid sequence
配列番号 17：合成配列 SEQ ID NO: 17: Synthetic sequence

Claims

請 求 の 範 囲  The scope of the claims
I . 配列番号 1 2で表されるアミノ酸配列を含むことを特徴とする、 不凍機能を 有する蛋白質またはべプチド。 I. A protein or a peptide having an antifreeze function, comprising the amino acid sequence represented by SEQ ID NO: 12.
2 . 配列番号 1 3で表されるアミノ酸配列を含むことを特徴とする、 不凍機能を 有する蛋白質またはべプチド。  2. A protein or peptide having an antifreeze function, comprising the amino acid sequence represented by SEQ ID NO: 13.
3 . 配列番号 1 2または 1 3で表されるァミノ配列が複数回繰り返されているァ ミノ酸配列を含む請求項 2に記載の蛋白質またはべプチド。  3. The protein or peptide according to claim 2, which comprises an amino acid sequence in which the amino sequence represented by SEQ ID NO: 12 or 13 is repeated a plurality of times.
4 . 次の（A)または（B)で表される蛋白質またはぺプチド。  4. Protein or peptide represented by the following (A) or (B):
(A)配列番号 4に記載のアミノ酸配列を有する蛋白質またはべプチド。  (A) a protein or peptide having the amino acid sequence of SEQ ID NO: 4;
(B)配列番号 4に記載のアミノ酸配列において、 1もしくは数個のアミノ酸残 基が欠失、 置換もしくは付加されたアミノ酸配列を含むアミノ酸配列からなり、 かつ、 不凍機能を有する蛋白質またはペプチド。  (B) a protein or peptide having an antifreeze function, which comprises an amino acid sequence containing an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4;
5 . 氷核蛋白質における繰り返しァミノ酸配列を含むアミノ酸配列からなる不凍 機能を有する蛋白質またはペプチド。  5. An antifreeze protein or peptide comprising an amino acid sequence containing a repeated amino acid sequence in an ice core protein.
6 . 氷核蛋白質を化学的または生化学的に部分分解することを含む、 請求の範囲 第 1項〜第 5項のいずれか 1項に記载の蛋白質またはぺプチドの製造方法。  6. The method for producing the protein or peptide according to any one of claims 1 to 5, which comprises partially decomposing the ice core protein chemically or biochemically.
7 . 請求の範囲第 1項〜第 5項のいずれか 1項に記載の蛋白質またはぺプチドを コードする DNA断片。  7. A DNA fragment encoding the protein or peptide according to any one of claims 1 to 5.
8 . 請求の範囲第 7項に記載の DNA断片を含むベクター。  8. A vector comprising the DNA fragment according to claim 7.
9 . ベクターが発現ベクターである請求の範囲第 8項に記載のベクター。  9. The vector according to claim 8, wherein the vector is an expression vector.
1 0 . 請求の範囲第 8項または第 9項に記載のベクターによって形質転換された 形質転換体。  10. A transformant transformed by the vector according to claim 8 or 9.
I I . 宿主が大腸菌である請求の範囲第 1 0項に記載の形質転換体。  I I. The transformant according to claim 10, wherein the host is Escherichia coli.
1 2 . 請求の範囲第 1 0項または第 1 1項に記載の形質転換体を培養して不凍機 能を有する蛋白質またはぺプチドを産生せしめ、 該蛋白質またはぺプチドを採取 することを特徴とする不凍機能を有する蛋白質またはべプチドの製造方法。  12. A method comprising culturing the transformant according to claim 10 or 11 to produce a protein or peptide having an antifreeze function, and collecting the protein or peptide. For producing a protein or a peptide having an antifreeze function.
1 3 . 蛋白質またはぺプチドをぺプチド合成機により化学的に合成することを特 徴とする請求の範囲第 1項〜第 5項のいずれか 1項に記載の蛋白質またはべプチ ドの製造方法。 13. The method for producing a protein or a peptide according to any one of claims 1 to 5, wherein the protein or the peptide is chemically synthesized by a peptide synthesizer. .
1 . 請求の範囲第 1項〜第 5項のいずれか 1項に記載の蛋白質またはペプチド を含むことを特徴とする氷再結晶防止剤または凝固点降下剤。 1. An anti-ice recrystallization agent or a freezing point depressant, comprising the protein or peptide according to any one of claims 1 to 5.
1 5 . 請求の範囲第 1項〜第 5項のいずれか 1項に記載の蛋白質またはペプチド を含むことを特徴とする氷スラリー。  15. An ice slurry comprising the protein or peptide according to any one of claims 1 to 5.
PCT/JP2004/001728 2003-02-17 2004-02-17 Antifreeze protein containing ice nucleating protein sequence WO2004072283A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005505033A JP4231928B2 (en) 2003-02-17 2004-02-17 Antifreeze protein containing sequence of ice nucleoprotein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003038751 2003-02-17
JP2003-038751 2003-02-17

Publications (1)

Publication Number Publication Date
WO2004072283A1 true WO2004072283A1 (en) 2004-08-26

Family

ID=32866404

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/001728 WO2004072283A1 (en) 2003-02-17 2004-02-17 Antifreeze protein containing ice nucleating protein sequence

Country Status (2)

Country Link
JP (1) JP4231928B2 (en)
WO (1) WO2004072283A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007105731A1 (en) * 2006-03-13 2007-09-20 Nippon Suisan Kaisha, Ltd. Protein having ice nucleating activity
CN110004161A (en) * 2019-03-20 2019-07-12 广东海洋大学 A kind of genetic engineering bacterium and its construction method for expressing high activity ice nucleation protein

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103965294A (en) * 2013-02-05 2014-08-06 中国科学院化学研究所 Antifreeze polypeptide, bionic antifreeze surface related to antifreeze polypeptide, and screening method and application of antifreeze polypeptide

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GRAETHER S P, ET AL: "beta-Helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect", NATURE, vol. 406, 2000, pages 325 - 328, XP002979633 *
GRAETHER S P, ET AL: "Modeling pseudomonas syringae ice-nucleation protein as a beta-helical protein", BIOPHYS J., vol. 80, 2001, pages 1169 - 1173, XP002979630 *
GREEN R L, ET AL: "Deletion mutagenesis of the ice nucleation gene from pseudomonas syringae S203", MOL. GEN. GENET., vol. 215, 1988, pages 165 - 172, XP002979632 *
LEINALA E K, ET AL: "A beta-helical antifreeze protein isoform with increased activity", J. BIOL. CHEM., vol. 277, 2002, pages 33349 - 33352, XP002979634 *
WARREN G, ET AL: "Conserved repeats in diverged ice nucleation genes from two species of pseudomonas", NUCLEIC ACIDS RES., vol. 14, 1986, pages 8047 - 8060, XP002979631 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007105731A1 (en) * 2006-03-13 2007-09-20 Nippon Suisan Kaisha, Ltd. Protein having ice nucleating activity
WO2007105734A1 (en) * 2006-03-13 2007-09-20 Nippon Suisan Kaisha, Ltd. Crustacean-derived protein having antifreeze activity
EP2006296A1 (en) * 2006-03-13 2008-12-24 Nippon Suisan Kaisha, Ltd. Crustacean-derived protein having antifreeze activity
EP2006295A2 (en) * 2006-03-13 2008-12-24 Nippon Suisan Kaisha, Ltd. Protein having ice nucleating activity
JPWO2007105731A1 (en) * 2006-03-13 2009-07-30 日本水産株式会社 Protein with ice nucleation activity
EP2006295A4 (en) * 2006-03-13 2010-03-24 Nippon Suisan Kaisha Ltd Protein having ice nucleating activity
EP2006296A4 (en) * 2006-03-13 2010-03-31 Nippon Suisan Kaisha Ltd Crustacean-derived protein having antifreeze activity
CN110004161A (en) * 2019-03-20 2019-07-12 广东海洋大学 A kind of genetic engineering bacterium and its construction method for expressing high activity ice nucleation protein
CN110004161B (en) * 2019-03-20 2022-09-13 广东海洋大学 Genetic engineering bacterium for expressing high-activity ice nucleoprotein and construction method thereof

Also Published As

Publication number Publication date
JPWO2004072283A1 (en) 2006-06-01
JP4231928B2 (en) 2009-03-04

Similar Documents

Publication Publication Date Title
Song et al. Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator
Bass et al. Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA
Wedaman et al. Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex.
US20120107875A1 (en) Expression Cassette, Recombinant Host Cell and Process for Producing a Target Protein
JP2008509682A (en) Protein production method using YEBF
WO2004072283A1 (en) Antifreeze protein containing ice nucleating protein sequence
JP3906357B2 (en) Multimerized high-performance antifreeze protein
US5928877A (en) Assay for an antifreeze protein
Solomon et al. Stable, High-Level Expression of a Type I Antifreeze Protein inEscherichia coli
KR100978758B1 (en) Ice-binding protein gene derived from Chaetoceros neogracile, recombinant vector comprising the gene, and protein encoded by the gene
LEROUX et al. Characterization of four new tcp-1-related cct genes from the nematode Caenorhabditis elegans
KR101492434B1 (en) Antifreeze Protein from Flavobacterium frigoris PS1
CN110577958B (en) Nucleic acid, recombinant plasmid, transformant, acetylcholinesterase and preparation method thereof
WO2004104201A1 (en) Antifreezing protein found in fish
JP2011229504A (en) Modified antifreeze protein improved in productivity and antifreeze activity and method for producing the same
Krauke et al. Chimeras between C. glabrata Cnh1 and S. cerevisiae Nha1 Na+/H+-antiporters are functional proteins increasing the salt tolerance of yeast cells
KR101080750B1 (en) Protein which confers cold-resistance
AU740755B2 (en) Fusion proteins comprising coiled-coil structures
Gulevsky et al. Ice-Nucleating Proteins of Bacterial Origin. Report II. Structure of Proteins and Their Genes
Vanderveer The function, characterization of expression, localization and activity of a divergent ice nucleating protein from Pseudomonas borealis
Saha et al. Regulation of late-acting operons by three transcription factors and a CRISPR-Cas component during Myxococcus xanthus development
McGill A molecular and phylogenetic analysis of cryobiosis in nematodes of the genus Panagrolaimus
Lin Intuition-based modeling and insights into how antifreeze proteins bind to ice
WO2018163947A1 (en) Protein crystallization utilizing porous protein crystal
Koepf Equine plasma gelsolin: from sequence to structure

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2005505033

Country of ref document: JP

122 Ep: pct application non-entry in european phase