JPH10127279A - Restriction enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new type II restriction enzyme specifically recognizing a new base sequence in a double stranded deoxyribonucleic acid, specifically cleaving the same and useful in a field of gene technology such as an analysis of a huge DNA by clouting a specific strain belonging to the genus Streptomyces. SOLUTION: This restriction enzyme is obtained by culturing Streptomeyces-sp. RH 232 (FERM-P-15725), etc., specifically recognized a base sequence of the formula (G is guanine; C is cytosine) in a double stranded deoxyribonucleic acid and specifically cleaves the same at a position marked by an arrow. The restriction enzyme Sse-232I produced by the Streptomyces-sp. RH 232, has the following physicochemical properties: (1) an optimum temperature is approximately at 37 deg.C; (2) an optimum pH range is 7.5-8.0; (3) molecular weight thereof is approximately 60,000-90,000 (by an equilibrium density gradient centrifugal separation method).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a type II restriction enzyme that specifically recognizes and cleaves a specific 8-base sequence in double-stranded deoxyribonucleic acid (DNA).

[0002]

2. Description of the Related Art Restriction enzymes are endo-nucleases capable of specifically recognizing and cleaving a specific DNA base sequence, and numerous restriction enzymes have been found.
Since the development of molecular genetics and biochemistry has revealed that DNA is the main body controlling genetics, restriction enzymes have been widely used at present, such as for elucidating genetic diseases and for genetic manipulation. Is a useful enzyme. Among them, it specifically recognizes DNA base sequence,
A type II restriction enzyme that cleaves is important and needed and has been used. To date, more than 300 types II restriction enzymes have been isolated, but combinations of DNA base sequences that cannot be recognized by these type II restriction enzymes still exist. That is, a new type II restriction enzyme is continuously required to further increase the chances of cutting DNA at a target position by an experimenter. Furthermore, when analyzing huge DNA as in various recent genome analysis projects, DN
It is desirable that the frequency of occurrence of the cleavage site of A is not so high, and for that purpose, a type II restriction enzyme (rare cutter) that specifically recognizes a DNA nucleotide sequence longer than 6 bases and specifically cleaves it is demanded. .

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel DNA base sequence capable of specifically recognizing and specifically cutting a novel DNA base sequence, and to be used in the field of genetic engineering such as analysis of huge DNA. It is to provide a useful novel type II restriction enzyme.

[0004]

SUMMARY OF THE INVENTION In summary of the present invention, the first invention of the present invention relates to a restriction enzyme, and comprises the following formula (1) in a double-stranded deoxyribonucleic acid:

[0005]

Embedded image

(Wherein G represents guanine and C represents cytosine), and has the ability to specifically recognize the base sequence represented by the following and to specifically cleave this at the position of the arrow. . Further, the second invention of the present invention relates to a method for producing the restriction enzyme of the first invention, and relates to Streptomyces (Streptomyces).
ces), wherein a strain that produces the restriction enzyme is cultured, and the restriction enzyme is collected from the culture.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. The type II restriction enzyme of the present invention may be any enzyme capable of specifically recognizing and cleaving the above base sequence, and these enzymes have the ability to produce a restriction enzyme that recognizes the eight bases of the double-stranded DNA. Strains, or mutants of these strains, or from these strains, a gene encoding this enzyme-producing ability was isolated using a conventional gene manipulation technique, and the recombinant was introduced into another organism. Can also be produced.

[0008] A specific example of a strain having the ability to produce an 8-base recognition restriction enzyme is Streptomyces sp. RH232. This fungus is a strain newly obtained by the present inventors from soil, and its microbiological properties are as shown in Table 1 below.

[0009]

TABLE 1 Cell wall type I type LL-diaminopimelic acid + meso - diaminopimelic acid - diaminobutyric acid - glycine + aspartic acid - ornithine - lysine - arabinose ^{* 1} - galactose ^{* 1} - quinone _{MK-9 (H 8),} MK-9 (H 6) Motosei presence of hyphae ^* 2 + Presence of aerial hyphae ^{* 2} + presence of spore linkage ^{* 2} + ───────────────────────────────────

* 1 Estimated using sulfuric acid hydrolyzate of all cells. * 2 Based on microscopic observation.

The analysis of cell wall components is described in the following three publications:
Japan Actinomycetes Study Group: “Experimental method for identification of actinomycetes”, (No. 1
(1985) Published by the Secretariat of the Japanese Society for Actinomycetes, edited by Kazuo Komagata: “Experimental Method for Chemical Classification of Microorganisms”, (First Edition)
2) Published by Gakkai Shuppan Center and co-authored by Eiko Yabuuchi and others:
"Bacterial identification method that accompanies a new taxonomy", (1st edition)
(1987) The cell wall type of the strain is type I according to the publication of Nane Publishing Co., Ltd. In the quinone analysis, menaquinone having a saturated hydrogen number of 6 or 8 in the multiprenyl side chain and 9 in the number of isoprene units in the isoprenoid side chain. Furthermore, the presence of aerial hyphae and spore chain were observed by morphological observation. From the above results, this strain was identified as an actinomycete belonging to the genus Streptomyces.

This strain is named and designated as Streptomyces sp. RH232, and has been deposited as FERM P-15725 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

[0013] Streptomyces sp. RH232
The 8-base recognition restriction enzyme produced by the strain has the above-mentioned enzymatic action and is named Sse232I.

The method for producing the restriction enzyme Sse232I according to the present invention will be described in more detail. First, at the time of culture, the nutrient source added to the medium is used by the strain used,
As long as it produces e232I, as a carbon source, for example, glucose, maltose, glycerin and the like can be used, and as a nitrogen source, yeast extract, peptone,
Corn steep liquor, meat extract, etc. are suitable. In addition, inorganic and metal salts such as phosphates, potassium salts, and magnesium salts may be added. Sse 232I
Although the production amount varies depending on the culture conditions, the culture temperature is generally 20 to 35 ° C., the pH of the medium is preferably 6 to 8, and the production of the restriction enzyme Sse232I reaches the highest in aeration and agitation culture for 1 to 3 days. Naturally, the culturing conditions are set so as to maximize the production amount according to the strain to be used, the composition of the medium, and the like. The restriction enzyme Sse232I produced by culturing the strain of the present invention mainly exists in the cells. Isolation of the cells from the culture solution can be performed, for example, by centrifugation. The extraction and purification of the present enzyme can be performed by a method according to a general restriction enzyme purification method. For example, cells are suspended in a buffer solution, crushed by sonication, and intracellular enzymes are extracted. Next, after removing cell debris by ultracentrifugation, for example, an extract of the obtained supernatant is salted out with ammonium sulfate. The resulting precipitate was dissolved in buffer A (10 mM potassium phosphate, pH 7.5, 10 mM 2-mercaptoethanol, 100 mM PMSF, 5% glycerin), dialyzed with the same buffer, and subjected to ion exchange chromatography.
Purification by a molecular sieve chromatography method, a hydrophobic chromatography method, an affinity chromatography method, or the like can be performed to obtain an enzyme preparation of the present restriction enzyme.

The method for measuring the activity of the restriction enzyme Sse232I is described below. After preheating 48 μl of a reaction solution having the composition shown in Table 2 below at 37 ° C., 2 μl of the present enzyme is added to bring the total volume to 50 μl, and the enzymatic reaction proceeds. After 10 minutes, 5 μl of an enzyme reaction stop solution (1% SDS, 50% glycerin, 0.02% bromophenol blue) is added to stop the reaction.

[0016]

TABLE 2 10 mM Tris-HCl pH 7.5 10 mM MgCl ₂ 7 mM 2-mercapto Ethanol 1.0 μg Ad-2 DNA

The reaction solution is overlaid on a 0.7% agarose slab gel and subjected to electrophoresis at a constant voltage of 10 V / cm for about 1 to 2 hours. The buffer for electrophoresis was 90 mM Tris-borate buffer (pH 8.3) 2.5 mM EDT
A is used. The DNA band can be detected by UV irradiation by previously containing 0.5 μg / ml ethidium bromide in the gel. The end point is when the number and amount of the DNA fragment bands no longer change.
Activity was defined as 1 μg of Ad-2 DNA per hour at 37 ° C.
Enzyme activity that completely cleaves [Adenovirus-2 DNA, manufactured by Bethesda Research Laboratories] is defined as one unit.

The restriction enzyme Sse232I has the following physicochemical properties.

(1) Action and substrate specificity The present enzyme is an enzyme that recognizes the base sequence represented by the formula (Formula 1) in a double-stranded DNA sequence and cleaves it at the position indicated by the arrow. The recognition site of the restriction enzyme Sse232I of the present invention was determined as follows. Restriction enzyme Sse232I
Cut Ad-2 DNA at two sites. However, λ-D
NA, pUC18 DNA, M13mp18 DNA, SV
40 DNA, ColEI DNA, pBR322DN
A, φX174 DNA was not cut. As a result, when the chain length of the obtained DNA fragment was searched, this enzyme was found to have the following formula (Formula 2) in the DNA sequence:

[0020]

## STR2 ## 5'-CGCCGGCG-3 '

It was suggested that they were aware of This 8
The base sequence contains a 6-base recognition restriction enzyme NaeI
[Formula (Formula 3)]:

[0022]

## STR3 ## 5'-GCCGGC-3 '

Containing Ad-2 DNA
Was further cleaved with NaseI and further cut with Sse232I, but there was no change in the pattern of the obtained DNA fragment, and it was concluded that the restriction enzyme Sse232I recognized the formula (Formula 2).

In order to determine the cleavage site of the restriction enzyme Sse232I, HindIII-SpeI of Ad-2 DNA having a recognition sequence of the enzyme and represented by SEQ ID NO: 1 in the sequence listing was used.
The fragment was Hind of M13mp18 (Takara Shuzo).
It was inserted into the dIII-XbaI site and used. From this plasmid for determination of cleavage, single-stranded D
Each NA was prepared. M13 represented by SEQ ID NO: 2 which binds to a site adjacent to the multiple cloning site
Primer M4 (manufactured by Takara Shuzo Co., Ltd.) was prepared by fluorescently labeling the 5 'end. The primers were annealed with single-stranded DNA prepared from the cleavage determination plasmid, respectively, and then Bacillus caldotenax was used.
tenax) DNA polymerase (BcaBEST DNA polymeras
e, manufactured by Takara Shuzo Co., Ltd.), a double-stranded DNA was cleaved with the present enzyme, and the chain length of the cleaved fragment was measured by denaturing polyacrylamide gel electrophoresis. At this time, the chain length obtained as a product is represented by the following formula (Formula 4):

[0025]

Embedded image

The band was detected as a band cleaved at the arrow indicated by the arrow, and was also detected by T4 DNA polymerase (T4 DNA Polymer).
merase (manufactured by Takara Shuzo Co., Ltd.), which was detected as a 4 bp long band, and concluded that the enzyme recognizes the base sequence represented by the formula (Formula 1) and cuts at the position indicated by the arrow. Was done.

(2) Optimal enzyme activity conditions I) Optimal temperature The optimal temperature of Sse232I was about 37 ° C. II) Optimum pH The optimum pH of Sse232I is in the range of pH 7.5 to 8.0. III) Salt concentration The optimal salt concentration of Sse232I is KCl or NaCl
In the case of, it was 0 mM. IV) MgCl ₂ concentration Sse232I has a MgCl ₂ concentration of 5 mM to ₂₀ mM.
The enzyme reaction was activated in the presence of.

(3) Determination of molecular weight The molecular weight was determined by an equilibrium density gradient centrifugation method. The density gradient was prepared by changing the glycerin concentration in a buffer having the composition shown in Table 3 below.

[0029]

Table 3 {10 mM Tris-HCl pH 7.5 10 mM 2-mercaptoethanol 30 mM KCl 10-25% glycerin} ───────────────────────

The bottom layer is 25 in two 5 ml centrifuge tubes.
%, And 4.8 ml of a linear density gradient liquid were prepared so that the uppermost layer was 10% glycerin. In addition, 1
The book was prepared by diluting 10 μl of the molecular range marker Low Range (manufactured by Bio-Rad) to 200 μl with the above buffer solution and overlaying a 10% glycerin layer, and the other was 18 μl of the Sse232I enzyme preparation. Was diluted in the same manner as above. These centrifuge tubes were centrifuged at 45000 rpm for 21 hours at 4 ° C. using a swing rotor. 25 centrifuge tubes in order from the upper layer after centrifugation
The buffer solution was withdrawn in an amount of 0 μl and fraction Nos.
20. The fraction of the molecular weight marker low range was subjected to SDS-PAGE, and the activity of the fraction of the enzyme preparation was measured. The peak of enzyme activity was detected in fraction No. 12, which was estimated to be about 60,000 to 90,000 in light of the molecular weight marker fraction. Therefore, the molecular weight of Sse232I is estimated to be about 60,000 to 90,000.

[0031]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of Column for Purification of Restriction Enzyme Sse232I Prior to enzyme purification, D for purification of restriction enzyme Sse232I was prepared.
An NA affinity column was prepared. First, a synthetic oligonucleotide having the base sequence shown in SEQ ID NO: 3 in the sequence listing, and a 5 ′ having the base sequence shown in SEQ ID NO: 4
Equivalent amounts of synthetic oligonucleotides whose terminal OH was labeled with biotin were mixed and annealed to obtain ligands. ImmunoPure ^oR Affin
Protocol attached to ityPak ^™ Immobilized Avidin [PIERCE] and literature [Paula J. Paula J.]
Science, Vol. 233, Vol. 1294
~ 1299 (1986)] with reference to ImmunoPure ^oR Aff.
The column was adsorbed to inityPak ^™ Immobilized Avidin to form a purification column for the restriction enzyme Sse232I.

(2) Purification of restriction enzyme Sse232I In a 100 ml Erlenmeyer flask, the medium 20 shown in Table 4 below was added.
ml, and 500 ml of the same medium were placed in four Erlenmeyer flasks each having a capacity of 2 liters, and the medium was sterilized by an ordinary method. A Streptomyces sp. RH232 (FERM P-15725) culture solution cultured with shaking at 30 ° C. for 48 hours in the medium charged in the 100-ml Erlenmeyer flask was added to the medium charged in the 2-liter Erlenmeyer flask in an amount of 5 ml each. And cultured at 30 ° C. for 27 hours. 42.7 g of cells were obtained from the culture by cooling and centrifugation.

[0034]

TABLE 4 Glycerin 17.5 g Polypeptone 3.5 g Meat extract 3.5 g Yeast extract 5g Food salt 2.0g Dipotassium hydrogen phosphate 1.0g Magnesium sulfate 0.5g Water water 1L ────────────────────────── ＰＨ pH 7.2 to 7.4

The obtained 42.7 g of cells were added to 130 ml of buffer A (20 mM Tris-HCl, pH 7.5, 1
0 mM 2-mercaptoethanol, 100 μM PM
SF) and crushed using an ultrasonic crusher.
The mixture was centrifuged at 000 × G for 1 hour to remove the residue, and 150 ml of extract was obtained. NaCl was added to the obtained supernatant at a final concentration of 0.3 M, and 30% ethyleneimine (polymer) (manufactured by Nacalai Tesque) diluted to 5% at a final concentration of 0.1%.
And stored on ice for 30 minutes. 2200
The mixture was centrifuged at 0 × G for 15 minutes, and ammonium sulfate (75 g) was added to the obtained supernatant to perform an ammonium sulfate precipitation operation.
The precipitate obtained by centrifugation at 22,000 × G for 15 minutes was mixed with buffer B (10 mM potassium phosphate buffer, pH
7.5, 10 mM 2-mercaptoethanol, 100
After dissolution in μM PMSF (5% glycerin), ammonium sulfate was added to a final concentration of 0.8M. Buffer B containing 0.8 M ammonium sulfate in advance
Adsorbed on a 50 ml column of Butyl Toyopearl (manufactured by Tosoh Corporation) equilibrated with the above, washed with buffer B containing 0.8 M ammonium sulfate, and then buffered with a linear concentration gradient of 0.8 to 0.3 M ammonium sulfate. Eluted with B. The obtained active fractions were combined and centrifuged CF2
5 (manufactured by Amicon). Buffer C (1
0 mM Tris-HCl, pH 7.5, 10 mM 2-
Mercaptoethanol, 50 mM KCl, 1 mM P
(MSF, 5% glycerin) for 4 hours, adsorbed to the 1 ml column prepared in (1) previously equilibrated with buffer C, washed sufficiently with buffer C, and then 0.05-0.8
Elution with buffer C with a linear gradient of 0 M KCl
A sample of this enzyme was obtained. This enzyme preparation contains non-specific DN
A-degrading enzyme and phosphatase were not contaminated.
According to the method described above, about 10 units of activity was obtained from 42.7 g of wet cells.

[0036]

As described in detail above, the present invention provides a restriction enzyme that recognizes and cuts the 8-base sequence of double-stranded DNA. The enzyme of the present invention is very useful in the field of genetic engineering for analyzing long-chain DNA and the like.

[0037]

[Sequence list]

Sequence number: 1 Sequence length: 755 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA sequence: AGCTTCCGAG GCCGAAGAGG TGTCAGACGA AACACCGTCA CCCTCGGTCG CATTCCCCTC 60 GCCGGCGCCC CAGAAATTGG CAACCGTTCC CAGCATCGCT ACA CTCCTCAGGC 120 GCCGCCGGCA CTGCCTGTTC GCCGACCCAA CCGTAGATGG GACACCACTG GAACCAGGGC 180 CGGTAAGTCT AAGCAGCCGC CGCCGTTAGC CCAAGAGCAA CAACAGCGCC AAGGCTACCG 240 CTCGTGGCGC GGGCACAAGA ACGCCATAGT TGCTTGCTTG CAAGACTGTG GGGGCAACAT 300 CTCCTTCGCC CGCCGCTTTC TTCTCTACCA TCACGGCGTG GCCTTCCCCC GTAACATCCT 360 GCATTACTAC CGTCATCTCT ACAGCCCCTA CTGCACCGGC GGCAGCGGCA GCGGCAGCAA 420 CAGCAGCGGT CACACAGAAG CAAAGGCGAC CGGATAGCAA GACTCTGACA AAGCCCAAGA 480 AATCCACAGC GGCGGCAGCA GCAGGAGGAG GAGCGCTGCG TCTGGCGCCC AACGAACCCG 540 TATCGACCCG CGAGCTTAGA AATAGGATTT TTCCCACTCT GTATGCTATA TTTCAACAAA 600 GCAGGGGCCA AGAACAAGAG CTGAAAATAA AAAACAGGTC TCTGCGCTCC CTCACCCGCA 660 GCTGCCTGTA TCACAAAAGC GAAGATCAGC TTCGGCGCAC GCTGGAAGACCGCGGG CTC 720 TCTTCAGCAA ATACTGCGCG CTGACTCTTA AGGAA 755

SEQ ID NO: 2 Sequence length: 24 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CGCCAGGGTT TTCCCAGTCA CGAC 24

SEQ ID NO: 3 Sequence length: 21 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CGAACGCGCC GGCGTTAGCT A 21

SEQ ID NO: 4 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TAGCTAACGC CGGCGCGTTC G 21

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd.

Claims (3)

[Claims] The present invention relates to a double-stranded deoxyribonucleic acid having the following formula: (Wherein G represents guanine and C represents cytosine). A restriction enzyme having the ability to specifically recognize a base sequence represented by the following formula and specifically cleave the base sequence at a position indicated by an arrow. 2. The restriction enzyme according to claim 1, wherein the restriction enzyme is a restriction enzyme Sse232I having the following physicochemical properties. (B) Optimum temperature: about 37 ° C (b) Optimum pH: pH 7.5 to 8.0 3. A method for producing a restriction enzyme, which comprises culturing a strain belonging to the genus Streptomyces and producing the restriction enzyme according to claim 1, and collecting the restriction enzyme according to claim 1 from the culture. Method.