JP5935382B2 - RrhJ1II 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 'or 3' end of the nucleic acid. On the other hand, an endonuclease is an enzyme that cleaves a nucleic acid inside the nucleic acid sequence (endo-), and a restriction enzyme is a typical endonuclease.

  Nucleases are useful enzymes that are currently widely used in genetic engineering techniques such as gene manipulation since it has become clear that DNA is the main body governing genes through the development of molecular biology or biochemistry.

  Nicking enzyme (Nicking enzyme) is an endonuclease that generates a nick in which only one strand of double-stranded DNA is cleaved, but it is used for nucleic acid amplification using its properties. Yes. 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, a method for isothermally amplifying a nucleic acid of 21 bases or more using a strand displacement DNA polymerase and a nicking enzyme has been developed (Patent Document 1).

  So far, more than 400 kinds of nicking enzymes have been reported, but only 4 cases of nicking enzymes have been reported in bacteria belonging to the genus Rhodococcus (Non-patent Document 2).

  The inventors have so far isolated a restriction enzyme and a corresponding modification enzyme from Rhodococcus rhodochrous J-1 strain, but this restriction enzyme has no nicking enzyme activity (Patent Document 2).

JP 2008-136451 A JP 2007-259853 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. Letek M, et al. , (2010) "The gene of a pathogenetic rhodococcus: cooperative viable underpinned by key gene acquisitions" PLO Genet. 2010 Sep 30; 6 (9).

  An 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 using the same.

  As a result of intensive studies to solve the above problems, the inventors isolated a novel nuclease RrhJ1II from a strain belonging to the genus Rhodococcus: Rhodococcus rhodochrous J-1.

That is, the present invention relates to a novel nuclease derived from a Rhodococcus microorganism.
The novel nuclease protein of the present invention is the following protein (A), (B) or (C).
(A) a protein comprising the amino acid sequence described in SEQ ID NO: 2 (B) an amino acid sequence described in SEQ ID NO: 2 comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having nicking enzyme activity (C) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 2 and having nicking enzyme activity

In addition, the novel nuclease protein has the nicking enzyme activity represented by the following formula 1 in double-stranded deoxyribonucleic acid:
(Wherein T represents thymine, G represents guanine, C represents cytosine, S represents guanine or cytosine, Y represents cytosine or thymine, and R represents adenine or guanine), and It may be a protein characterized by nicking enzyme activity for T / G mismatch in Formula 1.

The present invention also provides a gene encoding the novel nuclease protein.
Specifically, the novel nuclease gene of the present invention includes the following DNA (a), (b) or (c).
(A) DNA comprising the base sequence described 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 described in SEQ ID NO: 1 and encodes a protein having nicking enzyme activity
(C) DNA encoding a protein having a base sequence having a homology of 80% or more with DNA having the base sequence described in SEQ ID NO: 1 and having nicking enzyme activity

  The present invention also provides a recombinant vector containing the novel nuclease gene of the present invention, and a transformant containing the recombinant vector.

  Furthermore, the present invention also provides a method for producing the novel nuclease protein of the present invention. The production method can be carried out, for example, by culturing the above-described transformant and collecting a protein having nicking enzyme activity from the obtained culture. Preferably, the production method is performed using a cell-free protein synthesis method without using cells.

  According to the present invention, an endonuclease having nicking enzyme activity useful in the field of genetic engineering and a gene thereof are 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 type constant temperature 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 RrhJ1II expression plasmid pRR01. It is a figure which shows the result of the Western blotting using the His-tag specific antibody with respect to RrhJ1II protein refine | purified from recombinant Escherichia coli containing RrJ1II expression plasmid pRR01. It is a figure which shows the nuclease activity of RrhJ1II protein refine | purified from recombinant Escherichia coli containing RrhJ1II expression plasmid pRR01.

  Hereinafter, although an embodiment of the present invention is described in detail, this embodiment is an example for explaining the present invention, and the present invention is not limited only to this embodiment.

1. Novel nuclease of the present invention (RrhJ1II nuclease)
The novel nuclease of the present invention was first isolated by the inventors from Rhodococcus rhodochrous strain J-1. Rhodococcus rhodochrous J-1 strain (hereinafter also referred to as “J1 bacterium”) is deposited as FERM BP-1478 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki) It is a strain that has been.

  The inventors named this novel nuclease derived from J1 bacteria as “RrhJ1II”. In the present specification, the nicking enzyme activity possessed by the RrhJ1II nuclease is referred to as “RrhJ1II 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.

  In this specification, the nicking enzyme activity can be evaluated by bringing RrhJ1II nuclease into contact with DNA and measuring the molecular weight or the number of DNA fragments after the contact. A person skilled in the art can appropriately set conditions such as substrate DNA, 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 nicking enzyme activity can be evaluated by comparing the molecular weight of DNA before contact with the molecular weight of DNA after contact, or the number of DNA fragments before contact and the number of DNA fragments after contact.

  The inventors identified that RrhJ1II nuclease derived from J1 bacteria has the amino acid sequence shown in SEQ ID NO: 2.

  However, the RrhJ1II nuclease protein of the present invention is not limited to the one having the above sequence, and is about 50% or more, preferably about 60% or more, more preferably about 70% or more with the amino acid sequence shown in SEQ ID NO: 2. More preferably about 80% or more, particularly preferably about 90% or more, more particularly preferably about 95% or more, most preferably about 98% or more of an amino acid sequence having homology or identity, and nicking enzyme activity Proteins having the above are also included in the RrhJ1II nuclease of the present invention.

  In addition, the RrhJ1II nuclease protein of the present invention includes a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 2 and having nicking enzyme activity It is.

  Specifically, (i) an amino acid in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids have been deleted in the amino acid sequence described in SEQ ID NO: 2 Sequence, (ii) an amino acid in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids of the amino acid sequence shown in SEQ ID NO: 2 are substituted with other amino acids A sequence, (iii) an amino acid sequence in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are added to the amino acid sequence of SEQ ID NO: 2, (iv) An amino acid sequence in which 1 to 20 (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2) amino acids are inserted into the amino acid sequence shown in SEQ ID NO: 2, (v) (i) above ~ (Iv) combined Amino acid sequence, and the like.

  The amino acid substitution is preferably a conservative substitution between similar amino acid residues. For example, amino acids are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), sulfur Amino acids with atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), aromatic It is classified into amino acids (H, F, Y, W) having a containing side chain. 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 thereof 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 (RrhJ1II nuclease gene)
The RrhJ1II nuclease gene of the present invention is a gene encoding the aforementioned RrhJ1II nuclease protein. The RrhJ1II nuclease gene of the present invention includes, for example, DNA consisting of the base sequence set forth in SEQ ID NO: 1.

  The RrhJ1II nuclease gene of the present invention is not limited to the above sequence, but is about 50% or more, preferably about 60% or more, more preferably about 70% or more, and more preferably about 80 with the base sequence shown in SEQ ID NO: 1. % Or more, particularly preferably about 90% or more, more particularly preferably about 95% or more, and most preferably about 98% or more of DNA having a base sequence having a homology (identity) that also functions as a RrhJ1II nuclease. As long as it encodes a protein having (nicking enzyme activity), it is included in the gene of the present invention.

  Further, in response to the deletion, substitution or addition of the amino acid sequence described above, the base sequence described in SEQ ID NO: 1 has a base 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 RrhJ1II nuclease of the present invention, it is included in the RrhJ1II 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 may also be used as long as it encodes a protein having nicking enzyme activity. Included in genes.

  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, 4.41 g of sodium citrate dissolved in 1 liter of water), Hybridization by incubation with a probe at 65 ° C. for 20 hours in a solution containing 1% SDS, 100 μg / ml salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% ficoll. Although the conditions to perform can be mentioned, it is not necessarily limited to this. A person skilled in the art sets hybridization conditions in consideration of other conditions such as probe concentration, probe length, reaction time, etc. in addition to such conditions as salt concentration and temperature of the buffer solution. be able to.

  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.

  Hereinafter, an example of a method for obtaining the RrhJ1II nuclease gene by hybridization is shown, but the method is not limited thereto.

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 a suitable 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 to perform hybridization. As the probe, a polynucleotide encoding all or part of the amino acid sequence shown in SEQ ID NO: 2 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 RrhJ1II 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 RrhJ1II 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 RrhJ1II nuclease gene of the present invention as long as it is a DNA encoding a protein exhibiting the same activity as RrhJ1II.

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, such as QuikChange ^™ Site- Direct Mutagenesis Kit (Stratagene), GeneTailor ^™ Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System, etc.

  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. Maniatis et al., Molecular Cloning, A Laboratory Manual.

  Whether the obtained gene is a gene encoding the target RrhJ1II nuclease can be confirmed by comparing the determined base sequence with the base sequence described in SEQ ID NO: 1. 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.

3. Recombinant vector containing the novel nuclease gene of the present invention The present invention also provides a recombinant vector containing the RrhJ1II 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 is already present 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 this specification, the terminator can be, for example, a trp operon terminator, but is not limited thereto.

  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 target restriction enzyme or modification enzyme can be expressed after the introduction of the recombinant vector. Examples of the host include bacteria such as E. coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Rhodococcus, actinomycetes, yeast (Saccharomyces cerevisiae, Saccharomyces cerevisiae, Examples include animal cells, 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 RrhJ1II Nuclease of the Present Invention 5-1 Production by Cell Line The RrhJ1II nuclease of the present invention has an activity having characteristics (for example, nicking enzyme activity) of RrhJ1II nuclease from the culture after culturing the transformant. By collecting the protein, RrhJ1II 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, physically crushing it with a homogenizer, etc., and then centrifuging (15,000 rpm, 10 min, 4 ° C) to crush cells that cannot be disrupted (cell ) Can be obtained by collecting the supernatant. 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 RrhJ1II 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 combining known methods alone or appropriately. Also good. In this case, the enzyme activity, molecular weight, isoelectric point, etc. can be used for concentration and purification.

  For example, in collecting RrhJ1II nuclease from a culture of a transformant, after collecting the cells from the culture, the enzyme is extracted by ultrasonic disruption, ultracentrifugation, etc., and then the nucleic acid removal method, salting out method, What is necessary is just to refine | purify combining the affinity chromatography method, the gel filtration method, the ion exchange chromatography method etc. By this method, a large amount of RrhJ1II 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, RrJ1II nuclease can also be produced from the RrJ1II 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 or prokaryotic cell-derived extracts such as wheat germ, Escherichia coli and 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 RrhJ1II nuclease activity 6.1 Recognition sequence of RrhJ1II nuclease To examine the recognition sequence of RrhJ1II nuclease, for example, the host cell was transformed with a vector in which the gene shown in SEQ ID NO: 1 could be expressed by the method described above. Then, a protein having RrhJ1II nuclease activity is obtained from the culture. After the obtained protein was contacted with a sample for activity measurement by synthesizing double-stranded oligo DNA having a fluorescent label at the 5 ′ end of one strand and having various T / G mismatches in the sequence, When the DNA is denatured into a single strand by heating with a denaturant and subjected to SDS-polyacrylamide gel electrophoresis, detection by chemiluminescence is performed, and the cleavage pattern is analyzed, the recognition sequence of the J1 bacterium nuclease can be determined. . The RrhJ1II nuclease of the present invention has the following formula 1 in the following double-stranded deoxyribonucleic acid.
Wherein T is thymine, G is guanine, C is cytosine, S is guanine or cytosine, Y is cytosine or thymine, R is adenine or guanine, and The nicking enzyme activity for the T / G mismatch at the position indicated by the enclosed characters is shown. Here, “T / G mismatch” refers to a mismatched base pair in which a hydrogen bond is formed between thymidine (T) and guanine (G).

In Formula 1, the sequence of the portion represented by S, Y, R is not particularly limited as long as it is a base shown in the above description, but the T / G mismatch at the position indicated by the enclosed character is eliminated, and the lead chain It is preferable that a palindromic arrangement is obtained when T in the case becomes C. For example, the following formulas 2-4 can be mentioned.

  Hereinafter, the present invention will be described in more detail with reference to examples. The following are examples of the present invention and are not intended to limit the present invention.

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

  After the cultivation, 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, 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 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 RrhJ1II nuclease gene
Primers for amplifying the RrhJ1II nuclease gene by PCR were designed by the following method.

FIG. 1 shows the results of amino acid homology analysis of nucleases produced by Rhodococcus bacteria. The nucleases subjected to homology analysis in FIG. 1 and their origins are as follows.
A: DNA mismatch endoclease Vsr (Rhodococcus equi ATCC 33707)
B: NaeI very short patch repair endonuclease (Rhodococcus erythropolis SK121)
C: Very short patch repair protein (Rhodococcus jostii RHA1)
D: DNA missendonclease (Rhodococcus erythropolis PR4)
E: DNA missendonclease (Rhodococcus
opacus B4)

  In FIG. 1, two particularly conserved regions were selected and degenerate primers of SEQ ID NO: 3 and SEQ ID NO: 4 were designed, and degenerate PCR was 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 350b 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: 3) 1 μl
150 μM primer DG-02 (SEQ ID NO: 4) 1 μl
2.5 mM dNTP 8 μl
DMSO 10 μl
18 μl of sterilized water
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 of 30 cycles of reaction

Primer DG-01: 5'-GA (T / C) CCIGCIACITCIGCICGIATG-3 '(SEQ ID NO: 3)
DG-02: 5'-CCAIACICGIACIACICGTCCA-3 '(SEQ ID NO: 4)

  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: 5 was obtained. As a result of homology search, homology with the aforementioned endonuclease was confirmed.

[Example 3]
(1) Genomic Southern hybridization ApaLI, BamHI, ClaI, Eco52I, EcoT14I, KpnI, MluI, NcoI, NotI, PvuI, SacI, XbaI, XhoI When Southern hybridization was performed using the RrhJ1II probe prepared by the method, a single signal of about 1.2 kb was obtained from the fragment digested with Eco52I.

The probe of RrhJ1II 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 the AlkPhos Direct Labeling kit (GE Healthcare Bioscience) according to the attached manual, and used as a probe for RrhJ1II.

(2) Colony hybridization J1 bacterial genomic DNA was digested with restriction enzyme Eco52I, separated by 0.7% agarose gel electrophoresis, and GFX PCR DNA band and Gel Band Purification kit (GE Healthcare Biosciences) was removed from the gel. Used to recover about 1.2 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 / Eco52I cleavage fragment 4 μl
pBluescript II SK (+) / Eco52I 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 E. coli JM109 strain competent cell prepared by the method described below, and left 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. Then, 1 ml of SOC medium (20 mM glucose, 2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO4, 1 mM MgCl2) was added and cultured with shaking at 37 ° C. for 1 hour. . 200 μl of the culture solution after culturing 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 was obtained using the probe of RrhJ1II 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: 6 was obtained. The 450 bp open reading frame (ORF1) shown in SEQ ID NO: 2 was found in the base sequence shown in SEQ ID NO: 6. Since the amino acid sequence encoded by ORF1 has 87% to 89% homology with endonucleases derived from Rhodococcus bacteria of A to E shown in FIG. 1, ORF1 encodes an endonuclease. Therefore, the endonuclease encoded by ORF1 was named RrhJ1II nuclease. Further, the plasmid containing ORF1 obtained in Example 3 (2) was named pBRrhJ1II.

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 MgSO4, 1 mM MgCl2), and then at 18 ° C. for 20 hours. Cultured. 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 CaCl2, 40 mM MnCl2). 13 ml was added, allowed to stand 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, and the mixture was allowed to stand at 0 ° C. for 10 minutes, and then frozen using liquid nitrogen to obtain a competent cell.

[Example 4]
Production of RrhJ1II Nuclease by E. coli Recombinant (1) Construction of RrhJ1II Nuclease Expression Plasmid To obtain RrhJ1II 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 the RrhJ1II nuclease as a His-tag fusion protein, an SD sequence and a His-tag sequence were added upstream of the RrhJ1II nuclease gene.

Reaction solution composition
Template DNA (J1 bacterial chromosome DNA) 1 μl
2 × PCR Buffer KOD FX (Toyobo) 25 μl
10 μM primer N (SEQ ID NO: 7) 1.5 μl
10 μM primer C (SEQ ID NO: 8) 1.5 μl
2 mM dNTP 10 μl
1 μl of KOD FX DNA polymerase (Toyobo)
Total volume 50μl

Temperature cycle: 94 ° C: 120 seconds, 98 ° C: 10 seconds, and 68 ° C: 1 minute of reaction 30 cycles

Primer N: 5′-AGTGAATTCCCTTAAGAAGGAGATATACCATGCATCATCATCATCATCATCATGGCGTCGTCGGGAT-3 ′ (SEQ ID NO: 7)
C: 5′-GCCAAGCTTTCACCCCCGCGCCGGTTTT-3 ′ (SEQ ID NO: 8)

  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 0.5 kb was detected. After confirming the PCR product, the PCR product was purified from the reaction solution with 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 pRR01. FIG. 2 is a schematic diagram showing the structure of plasmid pRR01.

  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 recovered using Mini Plus Plasmid DNA Extraction kit (VIOGENE), EcoRI- The digestion with HindIII and sequence analysis were performed to confirm that the target DNA fragment was inserted.

(2) Preparation of purified enzyme The E. coli recombinant (JM109 / pRR01) containing the RrJ1II 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 100 ml × 1 LB liquid medium containing 100 μg / ml ampicillin and 1 mM IPTG, and cultured at 37 ° C. for 18 hours. The culture solution is collected by centrifugation, and the collected cells are suspended in a crushing buffer (composition 20 mM Sodium Phosphate, 0.5 M NaCl, 20 mM imidazole, 10% glycerol, pH 7.4) and then at 4 ° C. for 10 minutes. Ultrasonic crushing was performed. The disrupted solution was centrifuged, and the resulting supernatant was used as a cell-free extract. The cell-free extract was purified using a His-tag purification column (His Trap HP: GE Healthcare). The purified protein was eluted with Elution Buffer (0.5 M NaCl, 0.5 M imidazole, 20 mM Sodium Phosphate, 10% glycerol, pH 7.4), and fractionated into two fractions to be fraction 1 and fraction 2.

  The obtained purified sample was subjected to Western blotting using SDS-PAGE and His-tag specific antibody (GE Healthcare) to confirm that the target protein was purified. The results are shown in FIG. In FIG. 3, “1” in A and B is a molecular weight marker (BioRad Prestained Standard Broad), and “4” is a positive control (His × 6 fusion protein, 12 kDa). As a result of SDS-PAGE, a band appears in the vicinity of about 25 kDa in fraction 1 (FIG. 3A “2”) and fraction 2 (FIG. 3A “3”), and the target protein is obtained by Western blotting using a His-tag specific antibody. (Fig. 3B "2" "3").

(3) Measurement of enzyme activity Using the purified enzyme prepared in (2), RrhJ1II nuclease activity was examined. The activity was measured using a double-stranded oligo DNA having a biotin label at the 5 ′ end of one strand and having one T / G mismatch in the sequence.

Reaction solution composition
1.5 μM substrate DNA (SEQ ID NOs: 9 to 18) 2 μl
100 mM Tris-HCl (pH 7.5) 2 μl
100 mM MgCl ₂ 2 μl
1 mg / ml BSA 2 μl
Total volume 20μl

Reaction temperature: 20 ° C., 60 minutes

  After completion of the reaction, DNA was extracted with an equal amount of phenol to the reaction solution, heated at 100 ° C. for 2 minutes in the presence of formamide (final concentration 1 M), and then rapidly cooled on ice to denature the DNA into single strands. . The denatured single-stranded DNA sample was subjected to 20% polyacrylamide gel electrophoresis containing 8M urea, and chemiluminescence detection was performed using Imaging high chemikit (Toyobo) to compare the electrophoretic patterns.

  The results are shown in FIG. In FIG. 4, “1” is a 20-mer single-stranded DNA, “2” is a 40-mer single-stranded DNA, and “3” is a negative control (substrate DNA 01 that has been incubated and denatured in a reaction solution not containing an enzyme). It is. Oligonucleotides 02, 06, and 08 were cleaved to generate about 20-mer single strands (FIG. 4, “5”, “9”, and “11”). On the other hand, 01, 03, 04, 05, 07, 09 and 10 were not cut (FIG. 4, “4”, “6”, “7”, “8”, “10”, “12”, “13”. From these results, it was confirmed that RrhJ1II nuclease has an endonuclease activity that recognizes mismatches and cleaves DNA.

  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 3: Primer DG-01
Sequence number 4: Primer DG-02
Sequence number 7: Primer N
Sequence number 8: Primer C
SEQ ID NO: 9: Double-stranded oligo DNA01
SEQ ID NO: 10: Double-stranded oligo DNA02
Sequence number 11: Double stranded oligo DNA03
Sequence number 12: Double stranded oligo DNA04
SEQ ID NO: 13: Double-stranded oligo DNA 05
SEQ ID NO: 14: Double-stranded oligo DNA06
SEQ ID NO: 15: Double-stranded oligo DNA07
SEQ ID NO: 16: Double-stranded oligo DNA08
SEQ ID NO: 17: Double-stranded oligo DNA09
SEQ ID NO: 18: Double-stranded oligo DNA 10

Claims (7)

The following protein (A), (B) or (C),
(A) a protein comprising the amino acid sequence described in SEQ ID NO: 2 (B) an amino acid sequence described in SEQ ID NO: 2 comprising an amino acid sequence in which 1 or 10 amino acids have been deleted, substituted or added, and having nicking enzyme activity (C) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence described in SEQ ID NO: 2 and having nicking enzyme activity, provided that the nicking enzyme activity is represented by the following formula 1 in double-stranded deoxyribonucleic acid: :
(Wherein T represents thymine, G represents guanine, C represents cytosine, S represents guanine or cytosine, Y represents cytosine or thymine, and R represents adenine or guanine), and A protein characterized by nicking enzyme activity for the T / G mismatch in formula 1.   A gene encoding the protein according to claim 1. A gene comprising the following DNA (a) or (c):
(A) DNA comprising the base sequence described in SEQ ID NO: 1
(C) DNA encoding a protein consisting of a base sequence having 90 % or more identity with the DNA consisting of the base sequence described in SEQ ID NO: 1 and having nicking enzyme activity
However, the nicking enzyme activity is represented by the following formula 1 in double-stranded deoxyribonucleic acid:
(Wherein T represents thymine, G represents guanine, C represents cytosine, S represents guanine or cytosine, Y represents cytosine or thymine, and R represents adenine or guanine), and A gene encoding a protein, which is a nicking enzyme activity for the T / G mismatch in formula 1.   A recombinant vector comprising the gene according to claim 2 or 3.   A transformant comprising the recombinant vector according to claim 4.   A method for producing a protein comprising culturing the transformant according to claim 5 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 using a cell-free protein synthesis method, and collecting the protein having the nicking enzyme activity from a reaction solution.