JP6120066B2 - Novel nuclease and its gene - Google Patents

Description

  The present invention relates to a novel nuclease useful as a genetic engineering reagent, a gene encoding the enzyme protein, and a method for producing the enzyme.

  Nuclease is an enzyme that hydrolyzes a phosphodiester bond between a nucleic acid sugar and a phosphate into nucleotides. Depending on the type of degradation, it can be classified into endonuclease and exonuclease.

  Exonuclease degrades from the outside (exo-) of the nucleic acid sequence, ie, from the 5 'end or 3' end of the nucleic acid. On the other hand, endonuclease cleaves a nucleic acid inside the nucleic acid sequence (endo-).

  Two types of endonucleases are known, restriction enzymes and nicking enzymes. Restriction enzymes are useful enzymes that are currently widely used in genetic engineering technology.

  Nicking enzyme (Nicking enzyme or Nicking endonuclease) is an endonuclease that generates a nick in which only one strand of double-stranded DNA is cleaved. Since a normal nick (3'-hydroxy, 5'-phosphate) generated by a nicking enzyme serves as a starting point for various enzyme reactions, the nicking enzyme is used for DNA processing.

For example, a method is known in which a nick is formed on one side of a double-stranded DNA using a nicking enzyme, and a short nucleic acid of about 15 bases is isothermally amplified with a strand displacement DNA polymerase using the resulting nick as a priming site ( Non-patent document 1). As an improved technique of the method, a method of isothermally amplifying a nucleic acid having 21 bases or more using a strand displacement DNA polymerase and a nicking enzyme has been developed (Patent Document 1).
To date, more than 1000 kinds of nicking enzymes have been reported, but there are only 11 reports of bacteria belonging to the genus Rhodococcus, all of which are putative (http: //Rebase.neb.com/).

  The present inventors have so far isolated several nucleases from Rhodococcus rhodochrous J-1 strain and have found nicking enzymes that recognize mismatched sequences (Patent Documents 2 and 3).

JP 2008-136451 A JP 2007-259853 A JP 2013-005791 A

Jeffrey Van Ness, et al., (2003) “Isothermal reactions for the amplification of oligonucleotides” Proc. Natl. Acad. Sci. USA, Vol. 100, No. 8, 4504-4509

  Enzymes having various functions are required in the field of genetic engineering, and more types of enzymes having nicking activity than before are required.

  The main object of the present invention is to provide a novel nuclease and its gene useful in the field of genetic engineering, and to provide a method for producing the nuclease.

  As a result of intensive studies to solve the above problems, the inventors have isolated a novel nuclease (RrhJ1III) from a strain belonging to the genus Rhodococcus: Rhodococcus rhodochrous J-1.

The present invention is as follows.
The following protein (A), (B) or (C).
(A) a protein containing the amino acid sequence shown in SEQ ID NO: 2 (B) an amino acid sequence shown in SEQ ID NO: 2 containing an amino acid sequence in which one or several amino acids are deleted, substituted or added, and a nicking enzyme Protein having activity (C) A protein consisting of an amino acid sequence of 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having nicking enzyme activity

  According to the present invention, an endonuclease having nicking enzyme activity useful in the field of genetic engineering and a gene encoding the same can be provided. The novel nuclease of the present invention can be produced easily and in large quantities by recombinant production using the gene.

  Since the novel nuclease of the present invention has nicking enzyme activity, single-strand DNA cleavage for inducing homologous recombination for strand displacement isothermal nucleic acid amplification, visualization of chromosomal DNA by fluorescent labeling, and in vivo gene targeting It can be used for reactions.

It is a figure which shows the homology analysis result of the endonuclease which Rhodococcus genus bacteria produce. It is a schematic diagram which shows the structure of RrhJ1III expression plasmid pRR03. It is a figure which shows the enzyme activity of RrhJ1III.

(1) Novel nuclease (RrhJ1III nuclease) of the present invention
The novel nuclease according to the present invention was first isolated by the present inventors from Rhodococcus rhodochrous J-1 strain. Rhodococcus rhodochrous J-1 strain (hereinafter also referred to as “J1 fungus”) has been established as FERM BP-1478 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba City, Ibaraki) It has been deposited.

  The present inventors named this novel nuclease derived from J1 bacteria as “RrhJ1III”. The RrhJ1III nuclease has a nicking enzyme activity that turns the closed circuit of pBR322 into an open circuit. In the present specification, the nicking enzyme activity possessed by the RrhJ1III nuclease is referred to as “RrhJ1III nuclease activity”.

  In the present specification, the “endonuclease activity” means an enzyme activity that cleaves a nucleic acid inside the nucleic acid sequence (endo-), and the “nicking enzyme activity” means a double-stranded DNA having a specific sequence. It means an endonuclease activity that cleaves only one strand. The recognition site and cleavage site by the RrhJ1III nuclease may be the same or different.

  When the DNA cleaved by the RrhJ1III nuclease of the present invention is a double-stranded DNA, a nick is introduced into only one of the strands. Further, the DNA to be cleaved may or may not include a mismatch structure (for example, C / T mismatch) at the recognition site and the cleavage site.

  Nicking enzyme activity can be evaluated by contacting RrhJ1III nuclease with DNA and measuring the molecular weight or the number of DNA fragments after the contact. The nicking enzyme activity can be evaluated by comparing the molecular weight of DNA before contact with the molecular weight of DNA after contact, or by comparing the number of DNA fragments before contact with the number of DNA fragments after contact. . A person skilled in the art can appropriately set conditions such as DNA serving as a substrate, enzyme amount at the time of contact, temperature, solution composition, or contact time. The molecular weight of DNA can be measured, for example, by agarose electrophoresis.

  The present inventors have identified that RrhJ1III nuclease derived from J1 bacteria has the amino acid sequence shown in SEQ ID NO: 2 or 4. However, the RrhJ1III nuclease of the present invention is not limited to those having these sequences, and is not less than about 80%, preferably not less than about 85%, more preferably not less than about 80% of the amino acid sequence shown in SEQ ID NO: 2 or 4. A protein containing an amino acid sequence having homology or identity of 90% or more, particularly preferably about 95% or more, most preferably about 98% or more, and having nicking enzyme activity is also included in the RrhJ1III nuclease of the present invention.

  The RrJ1III nuclease of the present invention clearly has two types of nuclease (RrhJ1III nuclease) that differ only in the presence or absence of 3 amino acid residues near the N-terminus because the gene encoding the enzyme has two initiation codons. became. Both of these two nucleases are wild-type nucleases. In the present specification, when distinguishing between the two, the nuclease consisting of the amino acid sequence (SEQ ID NO: 2) translated from the first initiation codon is referred to as “RrhJ1III (1st)”. The nuclease consisting of the amino acid sequence (SEQ ID NO: 4) translated from the second start codon (next methionine) is referred to as “RrhJ1III (2nd)”.

  The RrhJ1III nuclease protein of the present invention includes an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and has nicking enzyme activity. Protein is also included.

  Specifically, (i) in the amino acid sequence shown in SEQ ID NO: 2 or 4, several, for example, 1 to 20 (preferably 1 to 10, preferably 1 to 5, more preferably 1 to 2) Amino acid sequence from which amino acids have been deleted, (ii) several amino acid sequences shown in SEQ ID NO: 2 or 4, for example 1 to 20 (preferably 1 to 10, more preferably 1 to 5, more (Preferably 1 to 2) amino acid sequence substituted with other amino acids, (iii) several, for example 1 to 20 (preferably 1 to 10) amino acid sequence shown in SEQ ID NO: 2 or 4 More preferably 1 to 5, more preferably 1 to 2 amino acid sequences added, (iv) several, for example 1 to 20 (preferably amino acid sequences shown in SEQ ID NO: 2 or 4) 1-10 , More preferably 1-5, more preferably amino acids are inserted in one to two), and the amino acid sequence of a combination of any of (v) above (i) ~ (iv).

  The amino acid substitution is preferably a conservative substitution between similar amino acid residues. Amino acids are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q) based on the properties of their side chains. , G, H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), a sulfur atom Amino acids (C, M) containing side chains, amino acids (D, N, E, Q) containing side chains containing carboxylic acids and amides, amino acids (R, K, H) containing side chains containing bases, aromatics It is classified into amino acids (H, F, Y, W) having side chains. It is known that the amino acids classified into each group are likely to maintain the activity of the polypeptide when substituted with each other, and such mutual substitution of amino acids is preferred. Examples include substitution between glycine and proline, glycine and alanine or valine, leucine and isoleucine, glutamic acid and glutamine, aspartic acid and asparagine, cysteine and threonine, threonine and serine or alanine, and lysine and arginine.

(2) Novel nuclease gene of the present invention (RrhJ1III nuclease gene)
The RrhJ1III nuclease gene of the present invention is a gene encoding the aforementioned RrhJ1III nuclease protein. The RrhJ1III nuclease gene of the present invention comprises DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3. The base sequence shown in SEQ ID NO: 1 is a gene encoding RrhJ1III (1st), and the base sequence shown in SEQ ID NO: 3 is a gene encoding RrhJ1III (2nd).

  The RrhJ1III nuclease gene of the present invention is not limited to the above sequence, and is about 80% or more, preferably about 85% or more, more preferably about 90% or more, more preferably the nucleotide sequence shown in SEQ ID NO: 1 or 3. DNA having a base sequence having a homology (identity) of about 95% or more, and most preferably about 98% or more is not limited as long as it encodes a protein having a function (Ricking enzyme activity) as an RrhJ1III nuclease. Included in the gene of the invention.

  Further, in response to the deletion, substitution or addition of the amino acid sequence described above, in the nucleotide sequence of SEQ ID NO: 1 or 3, a nucleotide sequence in which mutation such as deletion, substitution or addition has occurred in several bases, As long as it encodes a protein having a function (nicking enzyme activity) as the RrhJ1III nuclease of the present invention, it is included in the RrhJ1III nuclease gene of the present invention. The number of bases to be deleted, substituted or added is 30 or less, preferably 15 or less, particularly preferably 6 or less.

  Furthermore, a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3 may also be used as long as it encodes a protein having nicking enzyme activity. Included in the gene of the invention.

  Here, as stringent conditions, for example, a nylon membrane on which DNA is immobilized is 6 × SSC (1 × SSC is 8.76 g of sodium chloride and 4.41 g of sodium citrate dissolved in 1 liter of water. ) A solution containing 1% SDS, 100 μg / mL salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% ficoll (in this specification, “%” means “w / v And the conditions under which hybridization is carried out with a probe at 65 ° C. for 20 hours. However, the present invention is not limited to these conditions, and those skilled in the art can consider other conditions such as probe concentration, probe length, and reaction time in addition to conditions such as salt concentration and temperature of the buffer solution. Then, hybridization conditions can be set.

  For details of the hybridization procedure, see Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)) and the like can be referred to.

  An example of a method for obtaining the RrhJ1III nuclease gene by hybridization is shown below, but the present invention is not limited to this.

First, DNA obtained from an appropriate gene source is connected to a plasmid or phage vector according to a conventional method to prepare a DNA library. A transformant obtained by introducing this library into an appropriate host is cultured on a plate, and the grown colonies or plaques are transferred onto a nitrocellulose or nylon membrane, and after denaturation treatment, the DNA is immobilized on the membrane. This membrane is incubated in a solution having the above composition containing a probe previously labeled with ³² P or the like under the above stringent conditions, and hybridization is performed. As the probe, a polynucleotide encoding all or part of the amino acid sequence described in SEQ ID NO: 1 or 3 can be used.

  After completion of hybridization, the non-specifically adsorbed probe is washed away, and a clone that has hybridized with the probe is identified by autoradiography or the like. This operation is repeated until a hybridizing clone can be isolated. Finally, a gene encoding a protein having the target enzyme activity is selected from the obtained clones. The gene can be isolated by a known polynucleotide extraction method such as an alkali method.

  The RrhJ1III nuclease gene of the present invention can also be isolated from a microorganism that expresses the enzyme. For example, using a genomic DNA derived from Rhodococcus rhodochrous J-1 as a template, a primer or probe designed in consideration of gene degeneracy from known amino acid sequence information or a primer or probe designed based on known base sequence information The gene of interest can be isolated from the genome of the microorganism by the PCR or hybridization method used.

  The fact that various DNAs are included within the scope of the RrhJ1III nuclease gene of the present invention is derived from codon degeneracy. That is, it is known that there are 1 to 6 codons (a combination of three bases) for designating amino acids on a gene for each amino acid type. Therefore, there can be many genes encoding a certain amino acid sequence.

  Genes do not exist stably in nature, and it is not uncommon for mutations to occur in their base sequences. Depending on the mutation that has occurred on the gene, there is also a mutation that does not change the encoded amino acid sequence (called a silent mutation). In this case, it can be said that different genes encoding the same amino acid sequence have occurred. Therefore, even if a gene encoding a specific amino acid sequence is isolated, there is no denying the possibility that many kinds of genes encoding the same amino acid sequence will be created as the organism containing it is passaged. .

  Furthermore, it is not difficult to artificially create many kinds of genes that encode the same amino acid sequence using various genetic engineering techniques. For example, in the production of genetically engineered proteins, if the codons used on the original gene that encodes the target protein are infrequently used in the host, the protein expression level should be low. There is. In such a case, the target protein is highly expressed by artificially converting the codon to that frequently used in the host without changing the encoded amino acid sequence. Yes.

  Thus, it goes without saying that many kinds of genes encoding specific amino acid sequences can be artificially produced, and can also be generated in nature. Therefore, even if it is not the same gene as the nucleotide sequence disclosed in the present invention, it is included in the RrhJ1III nuclease gene of the present invention as long as it is a DNA encoding a protein exhibiting the same activity as RrhJ1III.

Methods for introducing mutations into genes and artificially modifying amino acid sequences include known methods such as the Kunkel method and Gapped duplex method, and mutation introduction kits using site-directed mutagenesis methods such as QuikChange ^™. Site-Directed Mutagenesis Kit (Stratagene), GeneTailor ^™ Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenism System (Mut-K, etc.) it can.

  Confirmation of the base sequence of DNA can be performed by sequencing by a conventional method. For example, a dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) can be used. It is also possible to analyze the sequence using an appropriate DNA sequencer.

  For the determination of the nucleotide sequence, in the case of a transformant prepared using a plasmid vector, if the host is Escherichia coli, culture is performed in a test tube or the like, and a plasmid is prepared according to a standard method. The obtained plasmid is used as a template as it is, or the inserted fragment is taken out and subcloned into an M13 phage vector or the like, and then the base sequence is determined by the dideoxy method. In the case of a transformant prepared with a phage vector, the base sequence can be determined by basically the same operation. The basic experimental method from the culture to the determination of the base sequence is described in, for example, the aforementioned T.C. In Maniatis et al., Molecular Cloning, A Laboratory Manual.

  Whether the obtained gene is a gene encoding the target RrhJ1III nuclease can be confirmed by comparing the determined base sequence with the base sequence described in SEQ ID NO: 1 or 3. Alternatively, the amino acid sequence deduced from the determined nucleotide sequence can be compared with the amino acid sequence described in SEQ ID NO: 2 or 4.

(3) Recombinant vector containing the novel nuclease gene of the present invention The present invention also provides a recombinant vector containing the RrhJ1III nuclease gene. The recombinant vector of the present invention can be produced by constructing an expression cassette by inserting a transcription promoter upstream of a gene encoding the above enzyme and optionally a terminator downstream, and inserting this cassette into the expression vector. it can. Alternatively, when a transcription promoter and / or terminator already exists in the expression vector, a gene encoding the enzyme is inserted between the promoter and / or terminator in the vector without constructing an expression cassette. do it.

  In the present specification, examples of the promoter include, but are not limited to, trc promoter, lac promoter, and the like. In the present specification, examples of the terminator include, but are not limited to, a trp operon terminator.

  In order to insert the gene of the present invention into a vector, a method using a restriction enzyme, a method using topoisomerase, or the like can be used. Further, if necessary at the time of insertion, an appropriate linker may be added. Ribosome binding sequences such as an SD sequence and a Kozak sequence are known as base sequences important for translation into amino acids, and these sequences can be inserted upstream of the gene. A part of the amino acid sequence encoded by the gene may be substituted with the insertion.

  The vector used in the present invention is not particularly limited as long as it retains the gene of the present invention, and a vector suitable for each host can be used. Examples of the vector include plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA and the like. For example, when Escherichia coli is used as a host, pTrc99A (GE Healthcare Bioscience), pACYC184 (Nippon Gene), pMW118 (Nippon Gene) and the like can be mentioned. Moreover, what modified these vectors can also be used as needed.

(4) Transformant Introducing the Vector of the Present Invention The transformant of the present invention can be produced by introducing the recombinant vector of the present invention into a host.

  The host used for the transformant is not particularly limited as long as the desired restriction enzyme or modification enzyme can be expressed after the introduction of the recombinant vector. Examples of the host include bacteria such as Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Rhodococcus, actinomycetes, yeast (Saccharomyces cerevisiae cells, Saccharomyces cerevisiae cells, Examples include plant cells and insect cells. In the present invention, the host is preferably E. coli.

  In the present invention, Escherichia coli is, for example, Escherichia coli K12 strain or B strain, or JM109 strain, XL1-Blue strain (for example, XL1-Blue MRF ′), a derivative strain derived from the wild strain, K802 strain, C600 strain, etc. Can be mentioned.

  The method of introducing the recombinant vector into the host is not particularly limited as long as it is a method suitable for the host, and those skilled in the art can appropriately select from known techniques. Examples of such a method include an electroporation method, a method using calcium ions, a spheroplast method, a lithium acetate method, a calcium phosphate method, and a lipofection method.

(5) Production method of RrhJ1III nuclease of the present invention (5-1) Production by cell line RrhJ1III nuclease of the present invention is obtained by culturing a transformant, and features of RrhJ1III nuclease from the culture (for example, nicking enzyme activity) By collecting a protein having an activity having RhJ1III nuclease, RrJ1III nuclease can be produced.

  In the present invention, the “culture” means a product obtained by culturing cells, a culture solution, a cell-free extract, a cell membrane and the like. The cell-free extract is prepared by adding the sodium phosphate buffer solution to the cell-free extract and physically crushing it with a homogenizer, etc., and then centrifuging (15,000 rpm, 10 min, 4 ° C.) to prevent crushing cells ( The supernatant can be recovered so that no cells are present. The cell membrane can be obtained as a pellet obtained by the above centrifugation, or can be obtained by suspending the pellet in a lysis buffer.

  As the RrhJ1III nuclease of the present invention, a culture may be used as it is, or a known method such as dialysis or ammonium sulfate precipitation, or a method obtained by concentrating and purifying from a culture by singly or appropriately combining known methods. Also good. In this case, the enzyme activity, molecular weight, isoelectric point, etc. can be used for concentration and purification.

  For example, in collecting RrhJ1III nuclease and its modified enzyme from a culture of a transformant, after collecting the cells from the culture, the enzyme is extracted by ultrasonic disruption, ultracentrifugation, etc. What is necessary is just to refine | purify combining the salting-out method, the affinity chromatography method, the gel filtration method, the ion exchange chromatography method etc. By this method, a large amount of RrhJ1III nuclease can be obtained.

  Depending on the expression system used, the enzyme protein expressed in the transformant may accumulate as an insoluble matter [inclusion body]. In this case, the insoluble matter is recovered, solubilized in a mild denaturing condition, for example, in the presence of a denaturing agent such as urea, and then the denaturing agent is removed to obtain an active protein. Furthermore, the target enzyme protein can be purified by performing the chromatography operation as described above.

(5-2) Production by cell-free protein synthesis system In the present invention, it is also possible to produce RrhJ1III nuclease from the RrJ1III nuclease gene of the present invention or the vector of the present invention using a cell-free protein synthesis system.

  The cell-free protein synthesis system is a system that synthesizes a protein in an artificial container such as a test tube using a cell extract. In other words, cell-free protein synthesis systems, unlike recombinant protein expression using living organisms, are a synthesis system that performs a series of protein synthesis flows such as transcription and translation in vitro without using cells. There is an advantage that a protein that becomes toxic can be produced. The cell-free protein synthesis system used in the present invention includes a cell-free transcription system that synthesizes RNA using DNA as a template.

  As the cell extract of the cell-free protein synthesis system, eukaryotic cell-derived or prokaryotic cell-derived extract, for example, wheat germ, Escherichia coli extract or the like can be used. These cell extracts may be concentrated or not concentrated.

The cell extract can be obtained by, for example, ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation or the like. Furthermore, in the present invention, cell-free protein synthesis can also be performed using a commercially available kit. Examples of such kits include reagent kits PROTEIOS ^™ (Toyobo), TNT ^™ System (Promega), synthesizer PG-Mate ^™ (Toyobo), RTS (Roche Diagnostics) and the like.

  Instead of these cell extracts, all the factors involved in protein synthesis are purified and reconstituted together with ribosomes, ATP, tRNA, amino acids, etc. It can also be used.

  Cell extracts used in normal cell-free protein synthesis systems consume a lot of energy other than protein synthesis due to coexistence of enzymes that hydrolyze ATP, resulting in low energy efficiency. Protein synthesis efficiency may be reduced by inhibitors such as proteases and nucleases.

  The “reconstituted cell-free protein synthesis system” does not have the problem of such an inhibitory factor, and can freely manipulate the constituent components, so that it is possible to design a system freely according to the purpose and target protein. . Further, the degree of freedom of the system is high, and the reaction system can be miniaturized or combined with an automation device to achieve high throughput.

  An example of “reconstructed cell-free protein synthesis system” is PURESYSTEM (registered trademark). PURESYSTEM (registered trademark) is reconstituted from E. coli, but the same system can be constructed using any of the other known cells, insect cells, wheat germ, and rabbit reticulocytes used in the cell-free protein synthesis system. Is possible.

  As described above, the nuclease of the present invention obtained by the cell-free protein synthesis system can be concentrated and purified by appropriately selecting chromatography as described above.

(6) Analysis of RrhJ1III nuclease activity To examine RrhJ1III nuclease activity, first culture J1 bacteria, collect the cultured cells and crush them by sonication, then collect the supernatant by ultracentrifugation. This is used as a sample for activity measurement. Further, as described above, a sample produced by a cell system or a cell-free protein synthesis system can also be used.

  After an appropriate amount of this sample is incubated at 37 ° C. with the substrate closed circular pBR322 DNA (pBR322), the degradation of the substrate DNA is confirmed by agarose gel electrophoresis.

  When the RrJ1III nuclease enzyme activity of J1 bacteria is examined using this method, the enzyme activity is detected as an activity that cleaves pBR322 into an open circuit. By analyzing the cleavage pattern by the RrhJ1III nuclease using various plasmids as substrate DNA, the recognition sequence of the RrhJ1III nuclease can be determined.

  Hereinafter, the present invention will be described in more detail with reference to examples. In this specification, “%” means “w / v”.

[Example 1]
Preparation of chromosomal DNA of J1 strain Rhodococcus rhodochrous J-1 strain was cultured with shaking in 100 mL of MYKG medium at 30 ° C. for 72 hours.

  After the culture, the cells were collected, and the collected cells were suspended in 4 mL of a Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl (pH 8.0)). After adding 40 mg of lysozyme to the suspension and shaking at 37 ° C. for 1-2 hours, the suspension was frozen at −20 ° C.

  Next, 10 mL of Tris-SDS solution (1% SDS, 0.1 M NaCl, 0.1 M Tris-HCl (pH 9.0)) was added with gentle shaking, and proteinase K (Merck) (10 mg / mL) was added. ) Was added and shaken at 37 ° C. for 1 hour.

  Next, an equal amount of TE (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) saturated phenol was added, and after stirring, centrifuged. After collecting the upper layer and adding twice the amount of ethanol, the DNA was wound up with a glass rod, and phenol was sequentially removed with 90%, 80%, and 70% ethanol.

  Next, the DNA was dissolved in 3 mL of TE buffer, ribonuclease A solution (100 ° C., heat-treated for 15 minutes) was added to 10 μg / mL, and the mixture was shaken at 37 ° C. for 30 minutes. Furthermore, after adding proteinase K and shaking at 37 ° C. for 30 minutes, an equal amount of TE saturated phenol was added and centrifuged to separate the upper layer and the lower layer.

  After repeating this operation twice for the upper layer, the same amount of chloroform (containing 4% isoamyl alcohol) was added, and the same extraction operation was repeated. Thereafter, twice the amount of ethanol was added to the upper layer, and the DNA was wound up and collected with a glass rod to obtain a chromosomal DNA preparation.

[Example 2]
PCR of the RrhJ1III nuclease gene
Primers for amplifying the RrhJ1III nuclease gene by PCR were designed by the following method.

  FIG. 1 shows the results of amino acid homology analysis of a putative nuclease produced by Rhodococcus bacteria. The nucleases subjected to homology analysis in FIG. 1 and their origins are as follows.

A: HNH endonuclease family protein (Rhodococcus sp. JVH1)
B: hypothetical protein W59_10709 (Rhodococcus imtechensis RKJ300)
C: hypothetical protein RHA1_ro03393 (Rhodococcus jostii RHA1)
D: hypothetical protein AK37_14191 (Rhodococcus pyridinivorans AK37)
In FIG. 1, two particularly conserved regions are selected and degenerate primers of SEQ ID NO: 5 and SEQ ID NO: 6 are designed, and degenerate PCR is performed under the following conditions using J1 bacterial genomic DNA prepared in Example 1 as a template. It carried out in. As a result, amplification of a band of about 280b was confirmed.

Reaction solution composition
Template DNA (J1 bacterial chromosomal DNA, Example 1) 1 μL
10 × Ex Buffer (Takara Bio Inc.) 10 μL
150 μM primer DG-01 (SEQ ID NO: 5) 1 μL
150 μM primer DG-02 (SEQ ID NO: 6) 1 μL
2.5 mM dNTP 8 μL
DMSO 10μL
Sterile water 18μL
ExTaq DNA polymerase (Takara Bio) 1 μL
Total volume 50μL

Temperature cycle: 94 ° C .: 30 seconds, 65 ° C .: 30 seconds and 72 ° C .: 1 minute reaction with 30 cycles primer DG-01: 5′-GGIAG (A / G) TGCTT (A / G) TGGGTG (C / T ) GGIAGG-3 ′ (SEQ ID NO: 5)
DG-02: 5'-CCT (T / C) AACTGICCGCT (T / C) AAGTA-3 '(SEQ ID NO: 6).

  Next, direct sequencing of the amplified sequence was performed using the primers used in PCR. As a result, the sequence shown in SEQ ID NO: 7 was obtained. As a result of homology search, homology with the above-mentioned endonuclease was confirmed.

[Example 3]
Cloning of RrhJ1III nuclease gene (1) Genomic Southern hybridization ApaLI, BamHI, ClaI, Eco52I, EcoT14I, KpnI, MluI, NcoI, NotI, PvuII, SalI, and XbaI were digested by the methods described below. When Southern hybridization was performed using the prepared RrhJ1III probe, a single signal of about 1.6 kb was obtained from the fragment digested with BamHI.

  The probe of RrhJ1III was prepared as follows.

  The PCR product prepared in Example 2 was purified using the GFX PCR DNA band and Gel Band Purification kit (GE Healthcare Bioscience). The purified PCR product was labeled using an AlkPhos Direct Labeling kit (GE Healthcare Bioscience) according to the attached manual, and used as a probe for RrhJ1III.

(2) Colony hybridization J1 bacterial genomic DNA was digested with restriction enzyme BamHI, separated by 0.7% agarose gel electrophoresis, and GFX PCR DNA band and Gel Band Purification kit (GE Healthcare Bioscience) was removed from the gel. Used to recover about 1.6 kb fragment. The obtained fragment was ligated to pBluescript II SK (+) vector (Stratagene) using a DNA ligation kit <Mighty mix> (Takara Bio). The reaction conditions are as follows.

Reaction solution composition:
Ligation light mix (manufactured by Takara Bio Inc.) 5 μL
J1 bacteria genomic DNA / BamHI fragment 4 μL
pBluescript II SK (+) / BamHI cleavage fragment 1 μL
Total volume 10μL
Reaction: 16 ° C., 1 hour.

The total amount of the ligation product was added to 200 μL of Escherichia coli JM109 strain competent cell prepared by the method described below and allowed to stand at 0 ° C. for 30 minutes. Subsequently, the competent cell was subjected to a heat shock at 42 ° C. for 45 seconds and cooled at 0 ° C. for 2 minutes. Thereafter, 1 mL of SOC medium (20 mM glucose, 2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO ₄ , 1 mM MgCl ₂ ) was added, and the mixture was incubated at 37 ° C. for 1 hour. Cultured with shaking.

  200 μL of the culture solution after the culture was applied to an LB AIX plate (LB agar medium containing 100 μg / L ampicillin, 100 μM IPTG, 50 μg / L X-gal) and left overnight at 37 ° C.

  94 white recombinant colonies grown on the plate were isolated on a new LB AIX plate for 10 plates per plate. Each plate was inoculated with 2 colonies / plate of JM109 strain transformed with pBluescript II SK (+) without insert.

  After the colony-isolated plate was left overnight at 37 ° C., the colony was copied onto a Hybond-N + (GE Healthcare Bioscience) membrane and colony high was obtained using the probe of RrhJ1III prepared in Example 3 (1). Hybridization was performed.

  A culture solution obtained by culturing the detected colonies was collected, and then the recombinant plasmid was recovered using QIAprep miniprep kit (manufactured by QIAGEN). Using a capillary DNA sequencer CEQ2000 (manufactured by Beckman Coulter), the base sequence of the genomic DNA fragment cloned in the plasmid was analyzed according to the attached manual.

  As a result, the base sequence represented by SEQ ID NO: 8 was obtained. In the nucleotide sequence shown in SEQ ID NO: 8, the 393 bp open reading frame (ORF1) shown in SEQ ID NO: 1 and the 384 bp open reading frame (ORF2) shown in SEQ ID NO: 3 were found. Since the amino acid sequences encoded by ORF1 and ORF2 have 68% to 94% homology to the endonucleases derived from Rhodococcus bacteria A to D shown in FIG. 1, ORF1 and ORF2 are endonucleases. Since it was presumed to encode a nuclease, the endonucleases encoded by ORF1 and ORF2 were designated as RrhJ1III nuclease (1st) and RrhJ1III nuclease (2nd), respectively. In addition, the plasmid containing ORF1 and ORF2 obtained in Example 3 (2) was named pBRrhJ1III.

  A competent cell of Escherichia coli JM109 strain was prepared by the following method.

E. coli strain JM109 was inoculated into 1 mL of LB medium and pre-cultured aerobically at 37 ° C. for 5 hours. Next, 0.4 mL of the preculture solution is added to 40 mL of SOB medium (2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO ₄ , 1 mM MgCl ₂ ) at 18 ° C. Cultured for 20 hours. The obtained culture was collected by centrifugation (3,700 × g, 10 minutes, 4 ° C.), and then cooled TF solution (20 mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl ₂ , 40 mM MnCl). ₂ ) 13 mL was added, left at 0 ° C. for 10 minutes, and centrifuged again (3,700 × g, 10 minutes, 4 ° C.) to remove the supernatant. The obtained Escherichia coli cells were suspended in 3.2 mL of a cold TF solution, 0.22 mL of dimethyl sulfoxide was added, the mixture was allowed to stand at 0 ° C. for 10 minutes, and then frozen with liquid nitrogen to obtain a competent cell.

[Example 4]
Production of RrhJ1III Nuclease by E. coli Recombinant (1) Construction of RrhJ1III Nuclease Expression Plasmid To obtain RrhJ1III nuclease, the J1 bacterial chromosomal DNA obtained in Example 1 was used as a template, and the reaction solution composition and primers shown below were used. PCR was performed. At this time, in order to express RrhJ1III nuclease as a His-tag fusion protein, an SD sequence and a His-tag sequence were added upstream of the RrhJ1III nuclease gene.

Reaction solution composition
Template DNA (J1 bacterial chromosome DNA) 3μL
2 × PrimeStar MaxDNA Polymerase (Takara Bio) 25 μL
10 μM primer N (SEQ ID NO: 9) 2 μL
10 μM primer C (SEQ ID NO: 10) 2 μL
Total volume 50μL
Temperature cycle: 98 ° C: 10 seconds, 55 ° C: 5 seconds and 72 ° C: 5 seconds of reaction 30 cycles

Primer N: 5'-GCGAATTCAAGGAGATATAGATATGCGTCACGATCCGGAGATG-3 '(SEQ ID NO: 9)
C: 5′-GTAAGCTTTCAATGGTGGATGGTGATGATCCTCACCACGCGTCGCCGCAACCCGCC-3 ′ (SEQ ID NO: 10).

  After completion of the reaction, 5 μL of the reaction solution was subjected to electrophoresis on a 1% agarose gel, and a PCR product of about 0.4 kb was detected. After confirming the PCR product, the PCR product was purified from the reaction solution using a DNA / RNA extension Kit (VIOGENE).

  The obtained PCR product was cleaved with restriction enzymes EcoRI and HindIII. The PCR product subjected to the restriction enzyme treatment was subjected to electrophoresis on a 1% agarose gel, and a band around about 0.5 kb was recovered. The collected PCR product was ligated to the EcoRI-HindIII site of the vector pUC18 to prepare a plasmid. The resulting plasmid was named pRR03. FIG. 2 is a schematic diagram showing the structure of plasmid pRR03.

  E. coli JM109 was transformed with this plasmid, and colonies were formed on an LB agar medium containing 100 μg / mL ampicillin, 1 mM IPTG, and 50 μg / mL X-gal. The obtained white colonies were inoculated into an LB liquid medium containing 100 μg / mL ampicillin, cultured overnight at 37 ° C., and then the plasmid was collected using Plasmid DNA Extraction Miniprep System (Viogene), and sequence analysis was performed. It was confirmed that the target DNA fragment was inserted.

(2) Confirmation of nuclease activity The E. coli recombinant (JM109 / pRR03) containing the RrJ1III nuclease expression vector prepared in (1) was cultured at 37 ° C. for 5 hours in 10 mL of LB liquid medium containing 100 μg / mL ampicillin. Was inoculated into 200 mL × 1 LB liquid medium containing 100 μg / mL ampicillin and 1 mM IPTG, and cultured at 25 ° C. and 150 rpm for 20 hours.

  The culture solution is collected by centrifugation, and the collected cells are suspended in a disruption buffer solution (composition: 20 mM Potassium phosphate (pH 7.4), 500 mM NaCl) and then disrupted by ultrasonic waves at 4 ° C. for 30 minutes ( Insulator (KUBOTA) 200W). The disrupted solution was centrifuged, and the resulting supernatant was used as a cell-free extract.

  The target protein was partially purified from the cell-free extract using a HisTrap column (Amersham Biosciences), and an enzyme reaction was performed at 37 ° C. in 20 μl of a reaction solution containing 0.5 μg of substrate DNA. As a result of separating the enzyme-treated plasmid DNA by agarose gel electrophoresis, it was shown that the closed circle pBR322 was changed to an open circle (FIG. 3).

  The novel nuclease and its gene of the present invention are useful in the field of genetic engineering, for inducing homologous recombination for strand displacement constant temperature nucleic acid amplification, visualization of chromosomal DNA by fluorescent labeling, and in vivo gene targeting. It can be used for single-strand DNA cleavage reaction.

Sequence number 5: Primer DG-01
Sequence number 6: Primer DG-02
Sequence number 7: His-tag primer HIS
SEQ ID NO: 8: Strept-tag primer STREP
SEQ ID NO: 9: transcription initiation signal primer (forward) NH
SEQ ID NO: 10: transcription initiation signal primer (reverse) CS

Claims (8)

The following protein (A), (B) or (C).
In the amino acid sequence shown in Protein (B) SEQ ID NO: 2 comprising the amino acid sequence shown in (A) SEQ ID NO: 2, 1-5 amino acids are deleted, substituted, looking contains additional or inserted amino acid sequence, and identity with the amino acid sequence shown in proteins (C) SEQ ID NO: 2 with a nicking enzyme activity Ri Do 95% or more amino acid sequences, and that having a nicking enzyme active protein The following protein (D), (E) or (F).
In the amino acid sequence shown in proteins (E) SEQ ID NO: 4 comprising the amino acid sequence shown in (D) SEQ ID NO: 4, 1-5 amino acids are deleted, substituted, looking contains additional or inserted amino acid sequence, and nicking amino acid sequence identity with shown in protein (F) SEQ ID NO: 4 having an enzymatic activity Ri Do 95% or more amino acid sequences, and that having a nicking enzyme active protein A gene comprising any of the following DNAs (a) to (d):
(A) DNA comprising the base sequence shown in SEQ ID NO: 1
(B) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 and encodes a protein having nicking enzyme activity
(C) the identity of the DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 is Ri Do 95% or more of the base sequence, and that encoding a protein having a nicking enzyme activity DNA
(D) DNA encoding a protein consisting of a base sequence of 1 to 15 bases substituted, deleted or added to the base sequence shown in SEQ ID NO: 1 and having nicking enzyme activity A gene comprising any of the following DNAs (e) to (h):
(E) DNA consisting of the base sequence shown in SEQ ID NO: 3
(F) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 3 and encodes a protein having nicking enzyme activity
(G) DNA and identity of the base sequence shown in SEQ ID NO: 3 is Ri Do 95% or more of the base sequence, and that encoding a protein having a nicking enzyme activity DNA
(H) DNA encoding a protein consisting of a base sequence of 1 to 15 bases substituted, deleted or added to the base sequence shown in SEQ ID NO: 3 and having nicking enzyme activity   A recombinant vector comprising the gene according to claim 3 or 4.   A transformant comprising the recombinant vector according to claim 5.   A method for producing a protein comprising culturing the transformant according to claim 6 and collecting a protein having nicking enzyme activity from the obtained culture.   A method for producing a protein, comprising synthesizing the protein having nicking enzyme activity according to claim 1 or 2 using a cell-free protein synthesis method, and collecting the protein having the nicking enzyme activity from a reaction solution.