JP2002325574A - Protein originating from extremely thermophilic bacterium and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To determine all sequence of a base of a genome of an extremely thermophilic bacterium Thermus thermophillus HB8 strain. SOLUTION: This gene provides a method producing a protein having a specific amino acid sequence originating from the bacterium, a gene having a base sequence encoding the protein, a vector including the same, a transformant, a protein obtained by culturing the transformant and collecting an expressed protein of the objective gene from culture product, for example, LexA protein known as a repressor protein, or a ferrochelatase which is an enzyme synthesizing protoheme from protoporphyrin IX or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thermophilic bacterium-derived protein and a gene encoding the same.

[0002]

2. Description of the Related Art The minimum number of basic genes required for life to survive is about 1,000. Although most of these genes are common to all organisms, including higher organisms, about one-third of them have completely unknown function.

[0003] At present, genome analysis of various organisms is underway, and analysis of the entire nucleotide sequence of some organisms including humans has already been made clear. At the same time, many new genes encoding proteins of unknown function have been discovered. All information for life is written in the base sequence of the genome. However, there is a limit to elucidating the functions of unknown proteins encoded by novel genes, and even the interactions between unknown proteins or known proteins, from only these nucleotide sequences. In order to elucidate all aspects of life phenomena and obtain industrially useful information from them, functional analysis of individual proteins will be absolutely necessary in the future.

A powerful weapon in elucidating the functions of unknown proteins is the knowledge obtained from the three-dimensional structure analysis of proteins. There are tens of thousands of protein types, but the structure is composed of thousands of basic units (folds)
It is considered to be a combination of Therefore, by solving the three-dimensional structure of all basic folds using known proteins and associating them with their functions, it is possible to clarify the functions of unknown proteins using these findings. Conceivable. For this purpose, it is desired to establish a large-scale three-dimensional structure analysis system for known proteins.

For the three-dimensional structure analysis, it is necessary to prepare a protein having excellent physical and chemical stability and easy to crystallize in a milligram order in a state of high purity. Proteins with excellent stability suitable for structural analysis can be obtained from thermophiles that can grow at 55 ° C or higher. In particular, a gene manipulation system has been established for the highly thermophilic bacterium Thermus thermophilus (T. th) which can grow even at 85 ° C., and it is possible to mass-produce proteins. Also, T.
Since th can grow in a minimal nutrient medium, it is considered that th has a minimum set of genes necessary for maintaining life. In addition, the minimum required gene structure is
It is not unique to T.th and is common to higher organisms including humans. Therefore, it is thought that life phenomena can be effectively elucidated by obtaining all the genes derived from Tth and analyzing the structure of the protein encoded by them. In fact, several T.th-derived genes have already been cloned, and the structure of the resulting protein has been solved. However, conventionally, these gene cloning
It uses probes and primers prepared from expression cloning or homologous sequences of other organisms using specific activity as an index, and requires time and effort. In order to carry out a large-scale three-dimensional analysis of proteins for the purpose of elucidating the full picture of life phenomena, it was necessary to clarify the overall picture of the T.th gene.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a protein derived from an extremely thermophilic bacterium and a gene encoding the same.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have determined the entire nucleotide sequence of the genome of the thermophilic bacterium Thermos thermophilus HB8 strain, Successfully identified 1137 genes contained in the present invention and completed the present invention.

That is, the present invention is as follows. (1) The following protein (a) or (b): (a) a protein containing any amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 2n (n represents an integer of 1 to 1137) (b) SEQ ID NO: 2n (n represents an integer of 1 to 1137) In one of the amino acid sequences selected from the amino acid sequence shown in 1 or a few amino acids is deleted, substituted or added, including a sequence, and a protein having a stability at 4 ℃ ~ 100 ℃ And microorganisms belonging to the genus Thermus (eg, Thermus thermophilus).

(2) A gene encoding the following protein (a) or (b): (a) a protein containing any amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 2n (n represents an integer of 1 to 1137) (b) SEQ ID NO: 2n (n represents an integer of 1 to 1137) A protein comprising a sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences selected from the amino acid sequences shown in (3) and having stability at 4 ° C to 100 ° C (3) or less A gene comprising the DNA of (c) or (d). (c) a DN consisting of any base sequence selected from the base sequences shown in SEQ ID NO: 2n-1 (n represents an integer of 1 to 1137)
A (d) DN consisting of any base sequence selected from the base sequences shown in SEQ ID NO: 2n-1 (n represents an integer of 1 to 1137)
DNA encoding a protein that hybridizes with A under stringent conditions and has stability at 4 ° C to 100 ° C

(4) A recombinant vector containing the gene. (5) A transformant containing the vector. (6) A method for producing the transformant, which comprises culturing the transformant and collecting an expressed protein of the target gene from the obtained culture. Hereinafter, the present invention will be described in detail.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a gene derived from a thermophilic bacterium and a protein encoded by the gene. These genes and proteins have excellent physicochemical properties such as pH stability, temperature stability, and stability to denaturing agents. In particular, it shows thermal stability such that the structure does not change even at around 70 ° C. Furthermore, it has high stability against denaturants such as urea. Hereinafter, the gene of the present invention will be described in detail.

1. Cloning of genomic DNA and genes Hyperthermophilic bacteria can grow in a temperature environment of 75-85 ° C.
Highly thermophilic bacteria inhabit the source of hot springs and the surrounding soil, etc., and are isolated by culturing an agar medium coated with a sample of hot water or soil from such a place at 70 ° C to 80 ° C. can do.

The highly thermophilic bacteria include microorganisms belonging to the genus Thermus, for example, Thermus thymophilus.
ermophilus, Thermus aq
uaticus). Next, genomic DNA is prepared from the thermophilic bacterium. Preparation of genomic DNA is a known method,
For example, it is prepared by a phenol-chloroform method or the like.

In the present invention, the DNA obtained by the above method can be analyzed by the whole genome shotgun sequence method. The whole genome shotgun sequencing method is to shred the whole genome, sequence it randomly and in large quantities, and then search for the end-to-end overlapping parts of each fragment using a computer and connect them.
This is a method to obtain sequence information of the whole genome. That is, the base sequence is determined for each DNA fragment treated with a restriction enzyme, or a DNA fragment fragmented at random positions by ultrasonic treatment or the like, and the sequence of a certain fragment is compared with the sequence of another fragment,
The overall sequence is determined by overlapping the overlapping parts.

The nucleotide sequence of the DNA fragment is determined by a known method, for example, the Sanger method (Molecular Cloning, Vol. 2, 13.3).
P., 1989), PCR-based methods and the like. Usually, the reaction is carried out using a PRISM sequencing kit containing a fluorescent dideoxy terminator manufactured by Perkin Elmer, etc., and the base sequence is determined using an auto sequencer (Model ABI 377) manufactured by Applied Biosystem.

Table 1 shows the thermophilic bacterium genes of the present invention.
Table 1 shows the nucleotide sequence and the amino acid sequence of each gene. These SEQ ID NOs correspond to the nucleotide sequence of the gene and the amino acid sequence of the general formula 2n-
"No." indicated by 1, 2n and described in the leftmost column of Table 1
Is applied to n (n represents an integer of 1 to 1137) in the above general formula. Thus, for example, the amino acid sequence of the LexA protein (# 2 in the table) is shown in SEQ ID NO: 4,
The nucleotide sequence of the LexA gene is shown in SEQ ID NO: 3.

[0017]

[Table 1]

However, as long as the protein containing the amino acid sequence encoded by the gene shown in Table 1 has stability at 4 ° C. to 100 ° C., a plurality of amino acids, preferably one or several Mutations such as deletion, substitution, and addition may occur. Furthermore, the above mutation may occur as long as the protein having the respective amino acid sequence has the intrinsic activity.

For example, 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 2n (n represents an integer of 1 to 1137) may be deleted. 1 to 10, preferably 1 to 5 amino acids may be added to the amino acid sequence shown in (n is the same as described above), or SEQ ID NO: 2n (n is 1 of the amino acid sequence shown in the above). Proteins of the present invention also include those in which 1 to 5, preferably 1 to 5, amino acids have been substituted with other amino acids.

Once the nucleotide sequence of the gene described in Table 1 has been determined, it is subsequently determined by chemical synthesis or
Using the base sequences at the 5 'and 3' ends as primers, genomic DNA
By PCR using all or part of
DN having the nucleotide sequence of the gene described in or the complementary sequence thereof
The respective genes can be obtained by hybridizing the A fragment as a probe. Alternatively, a mutant form of the gene of the present invention having the above activity can be synthesized by site-directed mutagenesis or the like. To introduce a mutation into a gene, the Kunkel method,
A known method such as the gapped duplex method or a method similar thereto can be employed. For example, a kit for mutagenesis using site-directed mutagenesis (for example, Mutan-K (TAKA
Mutations are introduced using RA) or Mutan-G (TAKARA).

The gene of the present invention also includes a gene that hybridizes with the above-mentioned DNA under stringent conditions and encodes a protein having the same function as the expression product of the DNA. Stringent conditions are:
This refers to conditions under which a specific hybrid is formed and a non-specific hybrid is not formed. For example, DNs having high homology (homology is 60% or more, preferably 80% or more)
The condition under which A hybridizes. More specifically,
The sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.

2. Preparation of Recombinant Vector and Transformant The recombinant vector of the present invention can be obtained by ligating the above gene to an appropriate vector, and the transformant expresses the recombinant vector of the present invention by expressing the target gene. It can be obtained by introducing it into a host as possible.

A phage or plasmid capable of autonomous propagation in a host microorganism is used as the vector. As the plasmid DNA, a plasmid derived from E. coli (for example, pBR3
22, pBR325, pUC118, pUC119, pUC18, pUC19, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.). Phage (Charon4
A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP
Etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.

To insert the gene of the present invention into a vector, first, the purified DNA is cut with an appropriate restriction enzyme,
For example, a method of inserting into an appropriate restriction enzyme site or a multiple cloning site of vector DNA and ligating to the vector is employed. The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exerted. Therefore, the vector of the present invention includes, in addition to the promoter and the gene of the present invention, cis elements such as enhancers, splicing signals, polyA addition signals, selection markers, ribosome binding sequences (SD sequences), if desired.
And so on. In addition, examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like. Furthermore, various shuttle vectors can be used in addition to vectors capable of autonomous propagation in two or more host microorganisms such as Escherichia coli and yeast. Such a vector can also be cleaved with the restriction enzyme to obtain a fragment thereof.

For linking the DNA fragment and the vector fragment, a known DNA ligase is used. Then, the DNA fragment and the vector fragment are annealed and then ligated to prepare a recombinant vector. The host used for transformation is not particularly limited as long as it can express the gene of the present invention. Examples include bacteria (such as Escherichia coli and Bacillus subtilis), yeast, animal cells (such as COS cells and CHO cells), and insect cells.

When a bacterium is used as a host, the recombinant vector of the present invention must be capable of autonomous replication in the bacterium and be composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Is preferred. Further, a gene controlling a promoter may be included. As Escherichia coli, for example, Escherichia coli (Esc
herichia coli) DH5α and the like, and as Bacillus subtilis, for example, Bacillus subtilis (Bacillus subtilis)
And the like. Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example trp promoter, lac promoter, P _L promoter, and P _R promoter may be used from Escherichia coli or phage. An artificially designed and modified promoter such as a tac promoter may be used. The method of introducing a recombinant vector into bacteria depends on the bacteria.
There is no particular limitation on the method for introducing DNA. For example, a method using calcium ions, an electroporation method and the like can be mentioned.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe
Are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast, for example, GAL1 promoter, GAL10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like. Can be used.
The method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

When an animal cell is used as a host, monkey cell CO
S-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells and the like are used. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like are used, and an early gene promoter of human cytomegalovirus may be used. Examples of a method for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method. When an insect cell is used as a host, an Sf9 cell or the like is used. Examples of a method for introducing a recombinant vector into insect cells include a calcium phosphate method, a lipofection method, and an electroporation method.

3. Production of useful substances, etc. In the present invention, utilizing the fact that highly thermophilic bacteria are stable in a high temperature region, the gene or protein of the present invention is used as basic data for analyzing the three-dimensional structure of all or a part of the protein. can do. Further, since the protein of the present invention is physicochemically stable, it can be used as a catalyst or the like in chemical industrial substance production.

In the present invention, the target protein (useful substance) can be obtained by culturing the transformant having the target gene, and collecting from the culture. The term “culture” means any of a culture supernatant, a cultured cell or a cultured cell, or a crushed cell or a cell. The method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host.

A culture medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be assimilated by the microorganism. If the medium can be performed efficiently,
Either a natural medium or a synthetic medium may be used.

As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As a nitrogen source, ammonia,
Ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, peptone, meat extract, corn steep liquor, and other nitrogen-containing compounds are used. As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

The cultivation is usually carried out at 37 ° C. for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture, the pH is kept near neutral. Adjustment of the pH is performed using an inorganic or organic acid, an alkaline solution or the like. During culture, antibiotics such as ampicillin and tetracycline may be added to the medium as needed.

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, isopropyl-β-D-thiogalactoside (IPTG) or the like is used.When culturing a microorganism transformed with an expression vector using the trp promoter, indoleacetic acid is used. (IAA) and the like may be added to the medium.

As a medium for culturing the transformant obtained using animal cells as a host, generally used RPMI16
For example, a 40-medium, a DMEM medium, or a medium obtained by adding fetal bovine serum or the like to such a medium is used. Culture is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed.

After the culture, when the target protein is produced in the cells or cells, the proteins are extracted by disrupting the cells or cells. When the target protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, common biochemical methods used for the isolation and purification of proteins, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography,
The target protein can be isolated and purified from the culture by using affinity chromatography or the like alone or in an appropriate combination.

Whether the target protein was obtained or not was determined by SD
It can be confirmed by S-polyacrylamide gel electrophoresis or the like. Furthermore, various tests can be performed to examine the physicochemical properties or functions of the target protein.
Test items include X-ray crystal analysis, CD spectrum analysis, N
MR analysis and the like.

4. Analysis of physicochemical properties (1) X-ray crystal structure analysis Crystallization of a target protein in the present invention can be performed according to a general crystallization method for X-ray structure analysis. For example, a precipitant may be added to a protein solution having a predetermined concentration to change the concentration, and the solubility of the molecule may be gradually lowered to precipitate as a crystal. Protein purity is important for crystallization, and it is essential to use a protein with high purity. Protein concentration is 5mg / ml
2020 mg / ml. Examples of the precipitant include ammonium sulfate, polyethylene glycol and the like.

The pH for crystallization is between 5.0 and 11.0,
The temperature is between 4C and 25C. PH is also an important parameter. Therefore, it is preferable to prepare a phase diagram in order to efficiently examine three parameters including pH (eg, precipitant, pH and concentration). Once the crystals have been deposited, the parameters are further refined to refine the conditions under which the best crystals appear. Here, it is preferable to equilibrate the sample during crystallization, in which case a vapor diffusion method can be employed.

In conducting a structural analysis in the present invention,
When the target protein has a high similarity to the structure of another protein, a molecular replacement method can be used. On the other hand, when the similarity with the structure of another protein is low, it is necessary to obtain the phase information of the crystal experimentally and perform the structural analysis. In this case, a method called a multi-wavelength anomalous dispersion method (hereinafter, referred to as a MAD method) that can be measured by using radiation that can change the wavelength of X-rays is employed. The MAD method is preferable in that the phase information can be obtained only with the heavy atom derivative, so that the structure can be determined even for a protein crystal whose crystal lattice is easily changed.

In order to introduce heavy atoms into a crystal, a permeation method is generally used. The infiltration method is an introduction method in which a crystal that has already grown is immersed in a solution containing a heavy atom reagent, and the property that the heavy atom reagent naturally penetrates into the crystal by diffusion is used.
Examples of heavy atom reagents include metals such as iridium compounds, mercury compounds, platinum compounds, uranium compounds, gold compounds, and lanthanoid compounds. When protein crystals are easily broken, sufficient time is required to prepare heavy atom derivatives of protein crystals.

In the present invention, the crystal structure analysis can be performed according to the so-called MAD method. In the present invention, the diffraction data of the crystal is measured by changing the incident X-ray wavelength using the emitted light. Synchrotron radiation that can be used for the MAD method is, for example, the large synchrotron radiation facility SPring-8 / RIKEN (RIKEN) beamline II.
(BL44B2). The wavelength range of the X-ray used is 1 ° to 2 °.

(2) CD spectrum analysis Among the steps from the purification of the protein of the present invention to the determination of the three-dimensional structure, a very important step is to determine whether or not the protein has the properties of the three-dimensional structure. It is to judge. This is because, even if expression and purification are performed smoothly, many proteins have an unnatural three-dimensional structure (mostly a random structure).

The CD (circular dichroism) spectrum can be easily inspected at a relatively low concentration (on the order of μM) to determine whether or not it has a tertiary structure (particularly a secondary structure). It is very useful as a measurement before the structure determination. The spectrum shows the difference in absorption coefficient (energy difference) of the amide bond to the left and right circularly polarized light with respect to the wavelength, and is usually in the far ultraviolet region (190-230n).
The secondary structure is determined from the absorption pattern of m). 20 helix-rich proteins as secondary structural elements
If the sheet has abundant negative maximum absorption at 8, 222 nm, positive and negative maximum absorption are observed on the spectrum at around 195, 215 nm, respectively. Further, by scanning only the wavelength of each maximum absorption with respect to the temperature change, the thermal stability of the protein can be observed. Generally, a sigmoid curve-like change is observed around the denaturation temperature, but if this slope is gentle, the tertiary structure of the protein is unstable and it can be determined that it has properties that are not suitable for X-ray and NMR analysis .

(3) NMR Spectrum Analysis Among the steps for determining the three-dimensional structure in the present invention, the means which can be used complementarily to the X-ray crystal structure analysis is NMR
(Nuclear magnetic resonance) method. As described above, not all purified proteins retain the three-dimensional structure, and may have a highly mobile loop region, or may be chemically modified other than polypeptide chains such as sugar chains. If so, crystallization is extremely difficult with membrane proteins.

On the other hand, the NMR method is a means capable of collecting three-dimensional structure information without crystallizing a sample. If the target protein is i) the molecular weight is 10 kDa or less, ii) 1
In the case of 0 to 30 kDa, iii) in the case of 30 kDa or more, or iv) in the case of fibrous or membrane protein, it is necessary to change the method of sample preparation and the method of spectral analysis. On the NMR spectrum, hydrogen atoms in the protein are observed at different frequencies (chemical shifts) depending on the environment. Utilizing this, the chemical shift of the hydrogen atom is separated in the 2-4 dimensional direction,
Distance information or angle information on the hydrogen atom of interest can be extracted.

In general, in the case of the above i), if the purified protein is replaced with a phosphate buffer or a deuterated buffer, information on the distance between ¹ H and angle is collected by the ¹ H multidimensional NMR method. be able to. Based on these three-dimensional structure information,
By performing molecular dynamics calculations with distance and angle restrictions,
Obtain the three-dimensional structure of the target protein. For ii), in order to prevent overlapping of the NMR signal, ¹³ C for ^{1 2} C, ¹⁴ N stable isotope present in the protein during sample preparation,
Must be replaced with ¹⁵ N. This means that a protein can be uniformly labeled by culturing a nitrogen source (mostly ammonium chloride) and a carbon source (mostly glucose) in the Escherichia coli minimum medium as the stable isotope. Triple resonance ( ¹ H, ¹³ C, ¹⁵
N) Multidimensional NMR spectrum measurement is performed, and a three-dimensional structure is obtained by molecular dynamics calculation from information on the distance between ¹ H and the angle as in i). In the case of iii) and iv), new means such as performing deuteration of side chain atoms by culturing in heavy water or obtaining orientation information of bonds such as NH and CH by orienting proteins are available. Required.

[0048]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. Example 1 Preparation of Genomic DNA Thermus thermophilus HB8 (ATCC 27634) was added to a Thermus nutrient medium (0.4% tryptone (DIFCO laboratories), 0.2%
Yeast extract (Oriental yeast), 0.1% NaCl, pH7.5) 5
After inoculating the cells in a ml and culturing at 70 ° C. for 15 hours, the cells were collected by centrifugation at 5,000 rpm for 10 minutes. The obtained cells were suspended in 500 μl of 10 mM Tris-HCl (pH 8.0) and 10 mM EDTA, and 10 mg of egg white lysozyme (Seikagaku Corporation) was added.
The cells were incubated for 20 minutes and lysed. RnaseA (Sigma) solution that has been heat-treated at 100 ° C for 10 minutes has a final concentration of 0.1 mg.
/ ml, and react at 37 ° C for 30 minutes.
μl of 10% SDS solution was added and incubated at 37 ° C. for 20 minutes. 1 mg of protease K was added and reacted at 50 ° C. for 30 minutes, and further incubated at 65 ° C. for 15 minutes. After performing phenol extraction, phenol-chloroform extraction, and chloroform extraction once each, the aqueous layer was overlaid with 1.5 ml of 100% cold ethanol, and the precipitated DNA was wound up with a Pasteur pipette having a rounded tip. 70% ethanol, 100
After rinsing the DNA with ethanol and drying, 200 μl of 10 mM Tris-HCl (pH 8.0), 10 mM EDTA overnight.
(TE) Dissolved in buffer.

[Example 2] Determination of base sequence of genomic DNA and detection / identification of gene 5 µg of genomic DNA obtained in Example 1 was added to 100 mM NaCl, 10 mM
MgCl ₂ , 1 mM dithiothreitol (DTT), 50 mM Tris-HCl
The digestion was carried out at 37 ° C. for 1 hour by using 0.1 unit of Sau3AI restriction enzyme in 50 μl of a buffer (pH 7.5). 1% of the reaction solution
The degree of progress of the restriction enzyme reaction was confirmed by agarose gel electrophoresis. The reaction solution was subjected to phenol extraction and ethanol precipitation to obtain a decomposition product.

The vector pUC18 (Amersham Pharmacia Biotech), which had been digested with the restriction enzyme BamHI and the terminal phosphate group was removed with bacterial alkaline phosphatase, and the above-mentioned Sau3AI digest of Thermus thermophilus HB8 genomic DNA were connected by ligation reaction, This reaction was used to transform E. coli JM109.
Plasmids were extracted and purified from the resulting transformants by a conventional method, and Thermus thermophilus contained in these plasmids was purified.
A sequence reaction was performed on the HB8 genomic DNA-derived sequence using Universal Primer (Takara Shuzo) and BigDyeKit (ABI). The obtained reaction product was analyzed using a 377A sequencer (ABI). Reading program Phr
The nucleotide sequence data obtained using ed (UWGC) is an assembly program PHRAP (UWGC: http: //www.genome.washingto
n.edu/UWGC/analysistools/phrap.htm). Genes were detected from the obtained base sequence of the whole genome using an ORF detection program (Looper or frame analyzer). BLAST (NCBI: http // ww
w.ncbi.nlm.nih.gov/BLAST/), PHRED (UWGC: http: // www.
genome.washington.edu/UWGC/analysistools/phred.ht
A homology search was performed using m). Table 1 summarizes the nucleotide sequence of each gene of the present invention and the amino acid sequence of the protein encoded by the gene.

Example 3 Thermus thermophilus HB8
Preparation of Derived LexA Gene Product In prokaryotes, there is an SOS response by LexA repressor protein as a mechanism corresponding to DNA abnormality. The SOS response is defined as the repressor protein LexA binding to a region called the SOS box located upstream of genes involved in DNA repair, replication, and cell division, and RecA bound to single-stranded DNA causes LexA to self-cleave. This is a response mechanism in which the expression of the suppressed gene is induced by promoting it (FIG. 1). In the case of Escherichia coli, the LexA protein is known to suppress 20 or more genes including the LexA gene itself. In this example, the LexA gene product was expressed and analyzed in order to isolate and express the LexA gene in Escherichia coli and to analyze the CD spectrum of the obtained expression product.

Using the following primers, PCR was performed using the Thermus thermophilus HB8 genomic DNA obtained in Example 1 as a template. 5 'primer: 5'-ATATCATATggCTCCCCCTTTgCCgACTggg
CAAAAgC-3 '(SEQ ID NO: 2275) 3' primer: 5'-ATATAgATCTTTATTAgTgCTCATAgCTCCA
CCTCAAAAgA-3 '(SEQ ID NO: 2276)

After boiling the reaction mixture for 10 minutes, 2.5 units of Taq DNA polymerase were added, and
Perform PCR using SystemPC-700 (Astec), and
The DNA fragment encoding xA was amplified. The amplification reaction conditions were 95 ° C for 3 minutes, then 95 ° C for 1 minute, 55 ° C for 3 minutes, 72 ° C
The 1 minute cycle was repeated 30 times, and finally the extension was completely stopped at 72 ° C. for 5 minutes. This PCR product and the vector pT7 Blue (No.
vagen) by the ligation reaction based on the principle of the TA cloning method, and the obtained recombinant vector pT7Blue-LexA
Was digested with 2 units of restriction enzyme NdeI and 1 unit of BglII at 37 ° C. for 1 hour in 40 μl of 100 mM NaCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, 50 mM Tris-HCl buffer (pH 7.5). Restriction enzymes NdeI, vector pET15b, which had been digested with BamHI and the terminal phosphate group had been removed with bacterial alkaline phosphatase, and the above-treated LexA fragment were connected by a ligation reaction, and the resulting recombinant vector pET15b-LexA was obtained. Was used to transform Escherichia coli BL21 (DE3) pLysE.

This transformant was transformed into an LBamp medium (1% bactotryptone, 0.5% yeast extract, 1% NaC
l, 50 μg / ml ampicillin) 2 ml, and cultured at 37 ° C for 16 hours. The obtained culture solution was added to 1 liter of LBamp medium, and 3
Incubate at 7 ° C for 3 to 4 hours.
0 μg / ml isopropyl-1-thio-β-D-galactoside (IPT
G) was added, and the cells were further cultured for 5 to 6 hours. The cells were collected by centrifugation, washed with TE, suspended in 20 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 0.2 M NaCl, 5 mM imidazole, and 1 mM 2-mercaptoethanol, and the cells were sonicated. Crushed. This crushed liquid was centrifuged at 10,000 g for 30 minutes to obtain a supernatant.

The histidine-tagged LexA protein in the obtained supernatant was adsorbed to chelating Sepharose. That is, after the nickel ions are bound, the cells are disrupted into chelating sepharose which has been sufficiently washed with an adsorption buffer (20 mM Tris-HCl (pH 8.0) containing 0.2 M NaCl, 5 mM imidazole, and 1 mM 2-mercaptoethanol). The supernatant of the solution was added and incubated at 4 ° C. for 1 hour. The Sepharose carrier was recovered by centrifugation and washed sufficiently with an adsorption buffer. 10 ml of an adsorption buffer containing 10 units of thrombin was added to this carrier, and the mixture was reacted at 37 ° C. for 1 hour to separate the histidine tag portion from LexA. After removing the carrier by centrifugation, the supernatant was applied to a benzamidine sepharose 6B column (Pharmacia) equilibrated with an adsorption buffer to adsorb LexA. 0.2M NaCl, 5mM EDTA
After thoroughly washing the column with 50 mM Tris-HCl buffer (pH 8.0) containing 50 mM Tris-HC1 containing 0.5 M NaCl and 5 mM EDTA
LexA was eluted with 1 buffer (pH 8.0). LexA purity
It was confirmed by 12.5% SDS polyacrylamide gel electrophoresis (FIG. 2). In addition, 50 mM Tris using Superdex200 HR
Gel filtration was performed using a buffer solution containing -HCl and 0.5 M KCl (pH 8.0), and the molecular weight was determined to be 39.8 kDa (Fig. 3).

Thermus thermophilus obtained as described above
2 μM of HB8-derived LexA protein, 50 mM containing 0.5 M NaCl
It was dissolved in a potassium phosphate buffer (pH 7.0) and subjected to CD spectrum analysis at 25 ° C. (FIG. 4). As a result, about 40
% Α-helix structure. In addition, in the same buffer, when the thermostability was examined, the structure of this LexA protein was 14 to 82 ° C, preferably 80 ° C.
Until stable (Fig. 4).

Example 4 Functional Analysis of LexA Gene (1) Self-Cleavage of LexA The state of self-cleavage of LexA in the presence and absence of RecA and ATPγS was analyzed by SDS-polyacrylamide gel electrophoresis (FIG. 5). . LexA in 50 mM Tris-HCl, pH 8.0, 50 μl
After 2 μg, 0.5 μg of RecA and ATPγS were added, and incubated at 37 ° C. for 0.5 or 3 hours, SDS-polyacrylamide gel electrophoresis was performed. When LexA undergoes self-cleavage, it produces the same two degradation products as those produced by alkaline treatment (lane 7). However, when incubated for 3 hours with ATPγS in the absence of RecA,
Almost no degradation products were observed (lane 3). Re
In the presence of cA and ATPγS, they are located at about 10 kDa and 15 kDa.
Two degradation products of LexA were observed (lanes 5 and
6). These reactions were significantly reduced in the absence of ATP (lane 4). These results indicated that LexA self-cleavage is promoted by RecA in the presence of ATP.

(2) Binding of LexA to Upstream Sequences of Putative SOS Gene The binding between LexA and the upstream sequences of four putative SOS genes was analyzed by a gel shaft assay (FIG. 6). Radiolabeled fragments derived from the upstream of each gene and Le
After incubation with xA, polyacrylamide gel electrophoresis was performed. As a result, in each case, a band having a lower electrophoresis than the labeled DNA fragment used in the reaction was confirmed. Therefore, it was shown that LexA binds to the above fragment.

(3) Binding to LexA Gene Upstream Sequence About 200 bp upstream of the LexA gene, binding to LexA was examined. This region was divided into about 50 bp. After incubating each fragment with LexA, polyacrylamide gel electrophoresis was performed, and the gel was stained with ethidium bromide and CBB. As a result, it was found that the mobility of the fragment in the region of -50 bp to -1 bp shifted to the high molecular side in the presence of LexA (FIG. 7). This band was also detected by CBB staining, indicating that the band was due to a complex of protein and DNA. From the above results, LexA is -50 bp upstream of LexA gene
Was specifically bound to the region.

Example 5 Preparation of Ferrochelatase Gene Product Ferrochelatase is an enzyme that is located at the final stage of the heme synthesis pathway and synthesizes protoheme by inserting Fe2 + into protoporphyrin IX in an ATP-independent manner (FIG. 8). ). It is known that deficiency of ferrochelatase causes myeloid protoporphyria in humans. Although few amino acids are completely conserved between organisms, histidine, which is thought to bind metal ions, is conserved in all ferrochelatase. From this, it is considered that the reaction mechanism and structure are widely conserved in eukaryotes and prokaryotes. Therefore, in this example, in order to examine the structure and function of ferrochelatase, ferrochelatase was prepared from a thermophilic bacterium, and a denaturation experiment, activity measurement, and crystallization were performed.

(1) Cloning of Ferrokeratase Gene The procedure of Example 1 was repeated, except that the following 5 ′ primer and 3 ′ primer were used.
Thermus thermophilus HB8 PCR using genomic DNA as template
Was done.

5'-side primer: 5'-ATATCATATGAACGTCCT
GCTCATGGCCTACGGCACCCCTTA-3 '(SEQ ID NO: 2277) 3' primer: 5'-ATATAGATCTTTATTATCTCGGCCACGCCGC
CTCCACCAGGTC-3 '(SEQ ID NO: 2278)

The PCR reaction conditions are the same as in Example 3. The PCR product and the vector pT7 Blue (Novagen) were connected by a ligation reaction based on the principle of the TA cloning method, and the obtained recombinant vector pT7Blue-FERR was added to 100 mM NaCl, 10 mM M
gCl ₂ , 1 mM dithiothreitol, 50 mM Tris-HCl buffer (pH 7.5) in 40 μl of restriction enzyme NdeI 2 units, BglII
Degraded by 1 unit at 37 ° C for 1 hour. Restriction enzyme NdeI,
Vector digested with BamHI and terminal phosphate group removed with bacterial alkaline phosphatase
pET15b and the fragment subjected to the above treatment were connected by a ligation reaction, and the obtained recombinant vector pE
Escherichia coli BL21 (DE3) pLysE was transformed using T15b-FERR.

(2) Purification of ferrochelatase The transformant obtained as described above was transformed into an LBamp medium (1% bactotryptone, 0.5% yeast extract, 1% NaC
l, 50 μg / ml ampicillin) 2 ml, and cultured at 37 ° C for 16 hours. The obtained culture solution was added to 1 liter of LBamp medium, and 3
Incubate at 7 ° C for 3 to 4 hours.
IPTG at 0 μg / ml was added, and the cells were further cultured for 5 to 6 hours. Collect the cells by centrifugation, wash with TE, then add 0.2M NaC
l, the cells were suspended in 20 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 5 mM imidazole and 1 mM 2-mercaptoethanol, and the cells were disrupted by sonication. 10,000 g of this crushed liquid
After centrifugation for 30 minutes, a supernatant was obtained. After the supernatant was heat-treated at 70 ° C. for 15 minutes, it was eluted with a 0 → 2 M NaCl gradient by affinity chromatography using Blue-Sepharose FF. Subsequently, ferrochelatase was purified by gel filtration using Sephacryl S-200. The purity of ferrochelatase was confirmed by 12.5% SDS polyacrylamide gel electrophoresis (FIG. 9).

[Example 7] Physicochemical properties of ferrochelatase In this example, to examine the structure and function of ferrochelatase that synthesizes protoheme from protoporphyrin IX,
A denaturation experiment, activity measurement, and crystallization were performed using ferrochelatase derived from the extreme thermophile Thermus thermophilus HB8.

(1) Determination of heat and pH 5 μM of ferrochelatase obtained as described above was added to 100 mM KC
dissolved in 50 mM potassium phosphate buffer (pH 8.0) containing
CD spectrum analysis was performed at 25 ° C to 100 ° C to examine the thermal stability. As a result, ferrochelatase is 25 ° C to 80 ° C
(Fig. 10, upper panel). Also, 100mM
The pH was set in the range of 2 to 12 using various buffers containing KCl, and the pH stability was examined. As a result, the ferrochelatase of the present invention was stable at pH 6 to 11 (lower panel in FIG. 10).

(2) Crystal Structure Analysis For more detailed analysis, the crystal structure of ferrokeratase was analyzed. The crystallization was performed by hanging drop vapor diffusion using polyethylene glycol and glycerol as precipitants. As a result, 10% PEG400
Crystallized successfully at 0, 10% glycerol, pH 6.5 (Fig. 1
1), 2.7Å resolution was obtained. Table 2 shows the crystallographic parameters, diffraction intensity data and electron density map.

[0068]

[Table 2] FIG. 12 shows a diffraction image, and FIG. 13 shows an electron density map.
Shown in

[0069]

According to the present invention, a thermophilic bacterium-derived protein and a gene encoding the same are provided. The protein of the present invention is useful as a reagent for analyzing a three-dimensional structure, and the gene of the present invention is useful as a raw material for mass production of a protein.

[0070]

[Sequence list]

[0071]

[Sequence List Free Text] SEQ ID NO: 2275: Synthetic DNA SEQ ID NO: 2276: Synthetic DNA SEQ ID NO: 2277: Synthetic DNA SEQ ID NO: 2278: Synthetic DNA

[Sequence list]

The sequence listing is published and registered in the Large Data CD-ROM "14
(2002) -002 (003) "

[Brief description of the drawings]

FIG. 1 is a diagram showing the function of LexA.

FIG. 2 shows the results of SDS-polyacrylamide gel electrophoresis of LexA.

FIG. 3 shows the results of LexA gel filtration.

FIG. 4 shows a CD spectrum and thermal stability of LexA.

FIG. 5 shows the self-cleavage of LexA.

FIG. 6 shows the binding of LexA to a putative SOS gene upstream sequence.

FIG. 7 is a diagram showing binding to a LexA gene upstream sequence.

FIG. 8 is a diagram showing a catalytic reaction of ferrochelatase.

FIG. 9 shows the results of SDS-polyacrylamide gel electrophoresis of ferrochelatase.

FIG. 10 is a graph showing test results of thermostability and pH stability of ferrochelatase.

FIG. 11 shows a crystal of ferrochelatase.

FIG. 12 is a view (X-ray diffraction image) showing the result of crystal analysis of ferrochelatase.

FIG. 13 is a view (electron density map) showing the result of crystal analysis of ferrochelatase.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/02 C 5/10 C12R 1:01 9/88 C12N 15/00 ZNAA C12P 21 / 02 A // (C12N 9/88 5/00 A C12R 1:01) (C12N 15/09 C12R 1:01) (C12P 21/02 C12R 1:01) F term (reference) 4B024 AA01 AA11 BA07 BA80 CA03 DA06 EA04 GA11 HA01 HA11 4B050 CC03 DD02 FF14 4B064 AG01 BA14 CA02 CA19 CC24 DA01 4B065 AA01Y AA26X AB01 AC02 BA22 CA24 CA27 4H045 AA10 CA11 DA89 FA74 GA26 HA05

Claims (8)

[Claims] 1. A protein of the following (a) or (b): (a) a protein containing any amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 2n (n represents an integer of 1 to 1137) (b) SEQ ID NO: 2n (n represents an integer of 1 to 1137) A protein having a sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences selected from the amino acid sequences shown in 4, and having a stability at 4 ° C to 100 ° C. 2. The protein according to claim 1, wherein the protein is derived from a microorganism belonging to the genus Thermus. 3. The microorganism belonging to the genus Thermus is S. cerevisiae.
3. The protein according to claim 2, which is a thermophilus. 4. A gene encoding the following protein (a) or (b): (a) a protein containing any amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 2n (n represents an integer of 1 to 1137) (b) SEQ ID NO: 2n (n represents an integer of 1 to 1137) A protein having a sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences selected from the amino acid sequences shown in 4, and having a stability at 4 ° C to 100 ° C. 5. A gene containing the following DNA (c) or (d): (c) a DN consisting of any base sequence selected from the base sequences shown in SEQ ID NO: 2n-1 (n represents an integer of 1 to 1137)
A (d) DN consisting of any base sequence selected from the base sequences shown in SEQ ID NO: 2n-1 (n represents an integer of 1 to 1137)
DNA encoding a protein that hybridizes with A under stringent conditions and has stability at 4 ° C to 100 ° C 6. A recombinant vector containing the gene according to claim 4 or 5. 7. A transformant comprising the vector according to claim 6. 8. A method for producing a protein, comprising culturing the transformant according to claim 6, and collecting an expressed protein of the target gene from the obtained culture.