JP2004236621A - Coat protein gene and phage vector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a heat-resistant protein in high efficiency by using a thermophilic phage. <P>SOLUTION: The method for the production of a heat-resistant protein contains (1) a step to integrate a gene encoding the heat-resistant protein into the DNA of a thermophilic phage, (2) a step to introduce the recombinant DNA obtained by the step 1 into a host cell and (3) a step to proliferate the thermophilic phage having the heat-resistant protein on a coat protein in the host cell. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】実施例で用いたリンカーを示す図である。
【図２】本発明のタンパク質を電気泳動で確認した結果を示す。
【図３】ファージに外来遺伝子を組み込み、コートタンパク質上に耐熱性タンパク質を発現する際の模式図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a thermophilic phage and a method for using the same.
[0002]
[Prior art]
A phage is generally composed of DNA (or RNA) that carries genetic information and a coat protein that wraps the DNA. Gene analysis of M13 and λ, which are cold phages that infect Escherichia coli, has progressed, and the gene sequence of the coat protein has been determined.
[0003]
However, φYS40, a phage derived from a thermophilic bacterium (for example, see Non-Patent Document 1),Thermus thermophilus HB27Although it is known to be infected, no genetic analysis has been done.
[0004]
In addition, thermophilic bacteria isolated by the inventor beforeThermus aquaticus TZ2Thermophilic phage φIN93 (see, for example, Patent Document 1) that infects bacteria has double-stranded circular DNA (about 19.6 Kbp), and the DNA is replicated in the cells and amplified as phage. I understand.
[0005]
Analysis of the gene sequence relating to the replication of the φIN93 double-stranded circular DNA has been advanced (for example, refer to Undisclosed Patent Document 1), and the promoter sequence has also been analyzed (for example, refer to Undisclosed Patent Document 2). However, information on coat proteins constituting phage particles has not been elucidated so far.
[0006]
In M13 of a room temperature phage that infects Escherichia coli, there has been reported an example in which a foreign gene is linked to a coat protein gene and expressed as a fusion protein on the surface of a phage particle (for example, see Non-Patent Document 2). However, thermophilic phages did not previously exist.
[0007]
[Non-patent document 1]
Sakiki, Y .; and Oshima, T .; Authors, "Isolation and Characterization of a Bacteriophage Infectious to an Extreme Thermophile, Thermos thermophilus HB8.", Journal of virology, Journal of virology. 1449-1453, 1975
[0008]
[Non-patent document 2]
Katsumi Maenaka and Masaru Furuta al., "A Stable Phage-Display System Using a Phagemid Vector: Phage Display of Hen Egg White Lysozyme (HEL), Escherichia coli Alkaline phosphatase, and Anti-HEL Monoclonal Antibody, HyHEL 10", Biochemical and Biophysical Rsearch Communications, 218, p. 682-687, 1996
[0009]
[Patent Document 1]
JP-A-7-67633
[0010]
[Undisclosed patent document 1]
Japanese Patent Application No. 2001-218934
[0011]
[Undisclosed patent document 2]
Japanese Patent Application 2001-201085
[0012]
[Problems to be solved by the invention]
A main object of the present invention is to efficiently produce a thermostable protein using a thermophilic phage.
[0013]
[Means for Solving the Problems]
The present inventors have found a method for expressing a thermostable protein on a coat protein of a thermophilic phage, and have completed the present invention.
[0014]
That is, the present invention relates to the following items 1 to 11.
[0015]
1. A thermophilic phage coat protein consisting of the following amino acid sequence (a) or (b);
(A) the amino acid sequence represented by SEQ ID NO: 2,
(B) an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the amino acid sequence (a), and which constitutes a thermophilic phage coat protein.
[0016]
2. A thermophilic phage coat protein consisting of the following amino acid sequence (c) or (d);
(C) an amino acid sequence represented by SEQ ID NO: 4,
(D) An amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the amino acid sequence (c), which constitutes a thermophilic phage coat protein.
[0017]
3. DNA consisting of the following (a), (b), (c) or (d);
(A) the gene sequence represented by SEQ ID NO: 1,
(B) a gene sequence encoding the amino acid sequence represented by (b) above,
(C) a DNA which hybridizes with the DNA consisting of the nucleotide sequence of (a) under stringent conditions and encodes a thermophilic phage coat protein;
(D) DNA which hybridizes with the DNA consisting of the nucleotide sequence of (b) under stringent conditions and encodes a thermophilic phage coat protein.
[0018]
4. DNA consisting of the following (e), (f), (g) or (h);
(E) the gene sequence represented by SEQ ID NO: 3,
(F) a gene sequence encoding the amino acid sequence represented by (d),
(G) a DNA which hybridizes with the DNA consisting of the nucleotide sequence of (v) under stringent conditions and encodes a thermophilic phage coat protein;
(H) DNA which hybridizes with the DNA consisting of the nucleotide sequence of (f) under stringent conditions and encodes a thermophilic phage coat protein.
[0019]
5. (1) a step of incorporating a gene encoding a thermostable protein into the DNA of a thermophilic phage, (2) a step of introducing the recombinant DNA obtained in the step (1) into a host cell,
(3) a step of growing a thermophilic phage in which a thermostable protein is expressed on a coat protein in the host cell;
[0020]
6. DNA of a thermophilic phage containing a gene encoding a thermostable protein to be expressed on a coat protein of the thermophilic phage.
[0021]
7. Item 7. A host cell containing the DNA according to Item 6.
[0022]
8. Item 6. A heat-resistant protein obtained by the method according to Item 5.
[0023]
9. Thermophilic phage expressing a thermostable protein on coat protein.
[0024]
10. (1) a step of incorporating a gene encoding a protein into the DNA of a thermophilic phage, (2) a step of mutating the recombinant DNA obtained in the step (1), and (3) a step obtained by the steps (2). Introducing a mutant DNA into a host cell, and (4) growing a thermophilic phage in which the protein is expressed on a coat protein in the host cell.
[0025]
11. Item 6. The method according to Item 5, wherein the thermophilic phage is φIN93.
[0026]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the thermophilic phage means a phage capable of infecting a thermophilic bacterium, and its type is not limited, and examples thereof include φIN93 and φYS40. The thermophilic bacterium refers to, for example, a bacterium that can grow at about 55 ° C. or higher, and its type is not limited. Examples of the thermophilic bacteria to which the thermophilic phage can infect include bacteria belonging to the genus Thermus, Bacillus, Clostridium, Pyrococcus, and the like.
[0027]
For example, when φIN93 is used as the phage, it is preferable to use a thermophilic bacterium, Thermus sp., As the host cell. The phage has about 19.6 Kbp of double-stranded bacterium DNA, and the DNA is replicated in the cells and amplified as phage.
[0028]
As bacteria of the genus Thermus, Thermus aquaticus (Thermus aquaticus TZ2 strain (FERM P-13713 deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary on June 29, 1993, Thermus aquaticus X-1 strain (Robert et al. (1950), Methods Med. Res., 2, 1-73), Thermus aquaticus strain YT-1 (J. Bacteriol. 98, 289-297, (1969), etc.), Thermus thermophilus, Thermus nonproteolies, and the like.
[0029]
The present inventor has for the first time found that the amino acid sequence of the coat protein in the thermophilic phage φIN93 (SEQ ID NO: 2 (coat protein A) and SEQ ID NO: 4 (coat protein B)) and the gene sequence encoding the protein (SEQ ID NO: 2) 1 (coat protein A) and SEQ ID NO: 3 coat protein (B)).
[0030]
Further, the present inventors introduce a gene encoding a thermostable protein (hereinafter, referred to as “foreign gene”) into the downstream portion of the gene encoding the coat protein, so that the thermostable phage has a thermostable phage on the coat protein. It has been found for the first time that a protein can be expressed.
[0031]
In the present invention, the coat protein of the thermophilic phage (hereinafter, also referred to as “the protein of the present invention”) is represented by SEQ ID NO: 2 or 4. Further, even a protein consisting of an amino acid sequence in which one or more, preferably one or several amino acids are substituted, deleted, inserted or added in SEQ ID NO: 2 or 4, may constitute a coat protein of a thermophilic phage. For example, it is included in the protein of the present invention.
[0032]
The present invention also includes a DNA encoding a coat protein of a thermophilic phage (hereinafter, sometimes referred to as “DNA of the present invention”). For example, (I) a DNA encoding the amino acid sequence represented by SEQ ID NO: 2 or 4 (preferably, a sequence represented by SEQ ID NO: 1 or 3, respectively); (II) an amino acid represented by SEQ ID NO: 2 or 4 A DNA encoding an amino acid sequence in which one or more, preferably one or several amino acids have been substituted, deleted, inserted or added in the sequence, and which constitutes a thermophilic phage coat protein, (III ) DNA that hybridizes with the DNA consisting of the base sequence represented by the above (I) under stringent conditions and encodes a coat protein of a thermophilic phage; (IV) the base sequence represented by the above (II) DNA that hybridizes under stringent conditions with DNA consisting of, and encodes a thermophilic phage coat protein. It is possible.
[0033]
Here, examples of the DNA that hybridizes with the above-described DNA under stringent conditions include the following:
(I) Under conditions of 50 ° C. in 0.2 × SSC containing 0.1% SDS or 60 ° C. in 1 × SSC containing 0.1% SDS, hybridizes with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1. DNA molecule having soy base sequence
(II) Hybridization with a DNA consisting of the nucleotide sequence of SEQ ID NO: 3 at 50 ° C. in 0.2 × SSC containing 0.1% SDS or at 60 ° C. in 1 × SSC containing 0.1% SDS DNA molecule having soy base sequence
The protein of the present invention can be obtained by a known biotechnological technique using the DNA of the present invention. For example, the DNA of the present invention can be amplified by a known method such as the PCR method to obtain a large number of copies (PCR products) of the gene sequence.
[0034]
Primers used in this PCR method can be appropriately set based on the sequence of the DNA, and can be synthesized by a known method. In addition, the amplified PCR product can be isolated, purified, and confirmed by a known method.
[0035]
The protein of the present invention can be obtained by a known gene recombination technique (for example, Science, 224, 1431, (1984); Bichem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983), etc.).
[0036]
For example, as for the protein of the present invention, a recombinant DNA (expression vector) capable of expressing a gene encoding the same in a host cell is prepared, and this is introduced into a host cell for transformation, and the transformant is cultured. Can then be recovered from the resulting culture.
[0037]
As the above host cells, both prokaryote and eukaryote can be used. As prokaryotic hosts, for example, commonly used ones such as Escherichia coli and Bacillus subtilis are widely used. Preferably E. coli, especially Escherichia. Coli-JM109 and the like.
[0038]
Eukaryotic host cells include cells such as vertebrates and yeast. As vertebrate cells, for example, monkey COS cells [Cell, 23: 175 (1981)], Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains [Proc. Natl. Acad. Sci. , USA. , 77: 4216 (1980)]. Saccharomyces yeast cells and the like can be suitably used as yeast cells. Of course, it is not limited to these.
[0039]
When a prokaryotic cell is used as the host, a promoter and an SD (Shine and Dalgarno) are used upstream of the gene so that the gene of the present invention can be expressed in the vector using a vector capable of replicating in the host cell. An expression plasmid to which a nucleic acid sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis have been added can be suitably used.
[0040]
As the above vector, a plasmid derived from Escherichia coli, for example, pET-21d, pBR322, pBR325, pUC12, pUC13 and the like are often used, but not limited thereto, and various known vectors can be used. Commercially available products of the above-mentioned vectors used in an expression system using Escherichia coli include, for example, pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMAl-P2 (New England Biolabs), pET21, pET21 / lacqen (Invitro). And pBAD / His (Invitrogen). Among these, pET-21d is particularly preferred.
[0041]
There is no particular limitation on the promoter, and when a genus Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter and the like can be preferably used. When the host is a bacterium belonging to the genus Bacillus, SP01 promoter, SP02 promoter, penP promoter and the like are preferable. When yeast is used as a host, for example, a pH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter and the like can be suitably used. When an animal cell is used as a host, preferable promoters include an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, and an SRα promoter.
[0042]
As an expression vector for the gene of the present invention, a normal fusion protein expression vector can also be preferably used. Specific examples of the vector include pGEX (Promega) for expression as a fusion protein with glutathione-S-transferase (GST).
[0043]
Examples of the nucleic acid sequence whose coding sequence of the mature polypeptide assists the expression and secretion of the polypeptide from a host cell include a secretory sequence and a leader sequence. These sequences can also be used for production of the protein (chitinase) of the present invention. In producing the protein of the present invention, a marker sequence (hexahistidine tag, histidine tag) used for purification of the fusion mature polypeptide with a bacterial host, and a hemagglutinin (HA) tag in the case of mammalian cells Etc. can also be used.
[0044]
A method for introducing a desired recombinant DNA (expression vector) into a host cell and a transformation method using the same are not particularly limited, and various general methods can be employed.
[0045]
The transformant can be cultured according to a conventional method, and the target protein (enzyme) of the present invention encoded by a gene designed as desired by the culture is expressed in the transformant, extracellularly, or on the cell membrane of the transformant. Produced (accumulated, secreted).
[0046]
As the medium used for the culture, various types of media commonly used depending on the host cells used can be appropriately selected and used. Culturing can also be performed under conditions suitable for the growth of the host cells.
[0047]
If desired, the recombinant protein of the present invention obtained as described above may be subjected to various separation operations utilizing its physical properties, chemical properties, etc. [“Biochemical Data Book II”, pp. 1175-1259, p. First edition, published on June 23, 1980 by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem. , 163, 313 (1987)].
[0048]
Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, ultrasonic crushing, ultrafiltration, molecular sieve chromatography (gel filtration). And various types of liquid chromatography such as adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof.
[0049]
Since the protein of the present invention is heat-resistant, it can be purified by heating. For example, a protein at room temperature is denatured and precipitated by heating, but a heat-resistant protein can be present in a supernatant because it does not denature. Using this technique, thermostable proteins can be easily separated.
[0050]
The protein of the present invention can also be obtained by chemical synthesis. As a chemical synthesis method, it can be produced by a known polypeptide synthesis method, for example, a liquid phase synthesis method or a solid phase synthesis method.
[0051]
Thus, the coat protein of the thermophilic phage of the present invention represented by SEQ ID NO: 2 or 4 can be obtained. As long as the protein of the present invention constitutes a coat protein of a thermostable phage, one or more, preferably one or several amino acids of these amino acid sequences may be deleted, substituted or added. .
[0052]
The protein of the present invention can be confirmed to be a coat protein by analyzing the N-terminal sequence after purification.
[0053]
In the present invention, a foreign gene can be incorporated into phage DNA by a conventional gene recombination technique using phage (for example, Scott, JM and Smith, GP, Science, 249, 386-390 (1990); Smith, GP and Scott, JK, Methods in Enzymology, 217, 228-257 (1993), etc.).
[0054]
The place where the foreign gene is incorporated in the DNA of the phage is not limited as long as the heat-resistant protein is expressed on the coat protein, and preferably includes a downstream portion of the DNA encoding the coat protein. When the thermophilic phage has a plurality of coat proteins, it may be incorporated into a downstream portion of the DNA encoding any coat protein.
[0055]
More specifically, for example, when φIN93 is used as a thermophilic phage, φIN93 has a coat protein (A) (SEQ ID NO: 2) and a coat protein (B) (SEQ ID NO: 4). A foreign gene may be incorporated into the downstream portion of the DNA encoding the protein.
[0056]
More preferably, a foreign gene can be incorporated into the “NruI site” located downstream of the DNA (SEQ ID NO: 1) encoding the coat protein (A). Thus, by incorporating a foreign gene into this portion, a heat-resistant protein can be expressed on the coat protein.
[0057]
There is no particular limitation on the foreign gene to be incorporated into the DNA of the thermophilic phage, and any gene can be used, and a gene encoding a thermostable protein is preferable. A gene encoding a thermostable protein refers to a protein (eg, an enzyme, an antibody, etc.) that can retain activity even at a temperature of 40 ° C. or higher, preferably 50 ° C. or higher, more preferably 60 ° C. or higher, and especially 70 ° C. or higher. Means the gene to be used.
[0058]
More specifically, a gene encoding a thermostable enzyme such as a thermostable kanamycin resistance gene, a thermostable β-glucosidase gene, a thermostable glutaminase gene, a thermostable protease gene, a gene encoding a heat shock protein, a groEL gene, groES Gene, dnaK gene, hsp70 gene and the like.
[0059]
The obtained recombinant DNA can be introduced (transfected) into a host cell (thermophilic bacterium) by a known method. For example, it is possible to cultivate thermophilic bacteria and the recombinant DNA of the present invention together (for example, see JP-A-2000-287686).
[0060]
By culturing the host cell thus obtained, a thermophilic phage in which a heat-resistant protein is expressed on a coat protein can be grown. Among the grown phages, a thermophilic phage expressing a desired heat-resistant protein can be purified, and the purified phage can be further infected to a host cell and grown.
[0061]
The method for purification is not particularly limited, and a commonly used method for purifying a protein can be used. For example, the antibody can be purified by immunoprecipitation, affinity chromatography, or the like using an antibody that specifically or selectively binds to the expressed thermostable protein.
[0062]
In this way, thermophilic phage expressing the desired thermostable protein can be produced in high purity and in large quantities. By separating and purifying the heat-resistant protein from the obtained phage by a known method, a desired heat-resistant protein can be obtained in a large amount with high purity (efficiently).
[0063]
In addition, an antibody that can specifically or selectively bind to a thermophilic phage, a host cell infected with the phage, and a thermostable protein can also be used as a biopanning kit.
[0064]
The present invention can also provide a method for improving protein heat resistance. More specifically, a gene encoding a protein that functions at room temperature can be incorporated into a downstream portion of a DNA encoding a coat protein of a thermophilic phage. The resulting recombinant DNA can then be mutated with a mutagen (eg, UV, nitrosoguanidine (NTG), acridine orange, ethyl methanesulfonate (EMS), etc.). Then, a thermophilic phage in which a heat-resistant protein is expressed on a coat protein can be obtained by introducing the mutant DNA into a host cell.
[0065]
In this way, a protein that exerts its effect at room temperature can be made heat-resistant. Further, by analyzing the DNA of the phage containing the protein whose temperature has been improved, the DNA sequence of the protein whose temperature has been improved can also be analyzed.
[0066]
【Example】
Examples are shown below to further clarify the features of the present invention.
[0067]
Example 1 : Thermophilic phage φ IN93 Of the gene sequence of the coat protein
Thermus aquaticus TZ2 FungusWas cultured overnight at 70 ° C. in an A2 medium (0.1% tryptone (Difco), 0.1% yeast extract (Difco) and Castenholtz basal salts pH 7.5), and 1 ml of the culture solution was added to 100 ml. The cells were re-inoculated into A2 medium and cultured at 70 ° C. in the same manner.
[0068]
At the stage when the turbidity was increased to OD = 0.2 (measured at a wavelength of 660 nm), the thermophilic phage φIN93 (1 × 10⁹(pfu / ml concentration) was added, and the cells were cultured at 70 ° C. for 3 to 4 hours. As a result, 2 × 10¹⁰Thermophilic phage φIN93 at a concentration of pfu / ml was confirmed.
[0069]
The culture solution was centrifuged at 3000 rpm for 10 minutes, and 100 ml of a PEG 8000 solution (polyethylene glycol (molecular weight 8000), 20%, 2M NaCl) was added to the supernatant from which the precipitated cell debris had been removed, and mixed. After standing for a period of time, the mixture was centrifuged at 10,000 rpm for 30 minutes, and the obtained phage was dissolved in 1 ml of distilled water and concentrated.
[0070]
Next, the phage concentrate was purified into only phage from which impurities such as bacterial cell debris were removed by density gradient centrifugation using cesium chloride (centrifugation at 55000 rpm for 15 hours). Cesium chloride was removed by dialysis with distilled water, and the purified phage was recovered as a final 1 ml solution.
[0071]
50 μl of the purified phage solution was boiled at 95 ° C. for 30 minutes. This sample was added to a 15% SDS polyacrylamide gel, and proteins were separated by electrophoresis. As a result of staining the proteins separated after the electrophoresis with Coomassie brilliant blue, two types of proteins were confirmed as coat proteins (FIG. 2).
[0072]
The separated two kinds of proteins were transferred to a nitrocellulose membrane by a transblot method, and amino acids from the N-terminal to the tenth residue were analyzed using an amino acid sequencer.
[0073]
As a result, with respect to the two types of coat proteins (A and B), the sequences of (A ') MADTAAIAAAQD and (B') MSENTLLGIAA, which are the N-terminal amino acid sequences, were obtained.
[0074]
The determined N-terminal amino acid sequence was converted into a DNA sequence, and searched using GENETYX (Hitachi Software) based on the entire base sequence of the DNA of φIN93. As a result, the positions of the genes corresponding to the two types of coat proteins (SEQ ID NOs: 2 and 4, respectively) were determined.
[0075]
The amino acid sequence of the obtained coat protein of the present invention and the DNA sequence encoding it are shown in SEQ ID NOs: 1 to 4.
[0076]
Example 2: Expression of heat-stable protein on coat protein
The NruI site of the restriction enzyme downstream of the coat protein gene (SEQ ID NO: 2) was cleaved. The GFP protein gene derived from the plasmid pGFP was digested with the restriction enzymes AgeI and EcoRI, and a ligase reaction was performed at 16 ° C. using the linkers (L1, L2) shown in FIG.
[0077]
DNA after reactionThermus aquaticus TZ2Bacteria were transfected (JP-A-2000-287686).Thermus aquaticus TZ2The bacteria were cultured overnight at 70 ° C. using A2 medium, and 50 μl of the culture was reinoculated into 5 ml of A2 medium and cultured at 70 ° C. in the same manner. At the stage where the turbidity was increased to OD = 0.2 (measured at a wavelength of 660 nm), 10 μl of the above-reacted DNA (total amount: about 100 ng) was added, and the cells were cultured at 70 ° C. for 12 hours.
[0078]
The above culture solution was centrifuged at 3000 rpm for 10 minutes, and the supernatant was separately cultured in A2 medium for 5 hours.Thermus aquaticus TZ2The bacteria were mixed with A2 medium containing about 1.0% agar kept at 50 ° C to 60 ° C, applied on an A2 medium plate containing 3% agar, and cultured at 70 ° C overnight to form plaque. Was.
[0079]
In the phage into which the GFP gene was introduced downstream of the coat protein gene of φIN93, the GFP protein was expressed on the coat protein and emitted green light.
[0080]
【The invention's effect】
With this expression system, it is possible to produce a large amount of heat-resistant protein that functions even at a high temperature of 70 ° C. or higher. Since the expressed protein is expressed on the coat protein of the phage, the expressed protein can be recovered in the same manner as the method for recovering the phage.
[0081]
In addition, it is possible to efficiently screen for an enzyme exhibiting a function at room temperature and having an increased temperature.
[0082]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a view showing a linker used in Examples.
FIG. 2 shows the results of confirming the protein of the present invention by electrophoresis.
FIG. 3 is a schematic diagram when a foreign gene is incorporated into a phage and a heat-resistant protein is expressed on a coat protein.

Claims (11)

A thermophilic phage coat protein consisting of the following amino acid sequence (a) or (b):
(A) the amino acid sequence represented by SEQ ID NO: 2,
(B) an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the amino acid sequence (a), and which constitutes a thermophilic phage coat protein. A thermophilic phage coat protein consisting of the following amino acid sequence (c) or (d);
(C) an amino acid sequence represented by SEQ ID NO: 4,
(D) An amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the amino acid sequence (c), which constitutes a thermophilic phage coat protein. DNA consisting of the following (a), (b), (c) or (d);
(A) the gene sequence represented by SEQ ID NO: 1,
(B) a gene sequence encoding the amino acid sequence represented by (b) above,
(C) a DNA which hybridizes with the DNA consisting of the nucleotide sequence of (a) under stringent conditions and encodes a thermophilic phage coat protein;
(D) DNA which hybridizes with the DNA consisting of the nucleotide sequence of (b) under stringent conditions and encodes a thermophilic phage coat protein. DNA consisting of the following (e), (f), (g) or (h);
(E) the gene sequence represented by SEQ ID NO: 3,
(F) a gene sequence encoding the amino acid sequence represented by (d),
(G) a DNA which hybridizes with the DNA consisting of the nucleotide sequence of (v) under stringent conditions and encodes a thermophilic phage coat protein;
(H) DNA which hybridizes with the DNA consisting of the nucleotide sequence of (f) under stringent conditions and encodes a thermophilic phage coat protein. (1) a step of incorporating a gene encoding a thermostable protein into the DNA of a thermophilic phage; (2) a step of introducing the recombinant DNA obtained in the step of (1) into a host cell; Growing a thermophilic phage in which the thermostable protein has been expressed on the coat protein in step (a). DNA of a thermophilic phage containing a gene encoding a thermostable protein to be expressed on a coat protein of the thermophilic phage. A host cell comprising the DNA according to claim 6. A heat-resistant protein obtained by the method according to claim 5. Thermophilic phage expressing a thermostable protein on coat protein. (1) a step of incorporating a gene encoding a protein into the DNA of a thermophilic phage, (2) a step of mutating the recombinant DNA obtained in the step (1), and (3) a step obtained by the steps (2). Introducing a mutant DNA into a host cell, and (4) growing a thermophilic phage in which the protein is expressed on a coat protein in the host cell. The method according to claim 5, wherein the thermophilic phage is φIN93.

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