JP3498806B2 - New restriction enzyme - Google Patents

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 7-base sequence in a double-stranded deoxyribonucleic acid (DNA).

[0002]

2. Description of the Related Art A restriction enzyme is an endo-type nuclease capable of specifically recognizing and cleaving a specific DNA base sequence, and many restriction enzymes have been found.
Since the development of molecular genetics and biochemistry has revealed that DNA is the main body that controls heredity, restriction enzymes are now widely used for elucidating genetic diseases and for genetic engineering. It is a useful enzyme. Of these, the DNA base sequence is specifically recognized and the DNA is specifically recognized.
Type II restriction enzymes, which cleave, are important and needed. To date, more than 300 types II restriction enzymes have been isolated, but there are still combinations of DNA base sequences that cannot be recognized by these type II restriction enzymes. That is, in order to further increase the chances of the experimenter to cleave DNA at a desired position, a new type II restriction enzyme is continuously required. Furthermore, when analyzing a huge DNA such as various genome analysis projects in recent years, DN
It is desirable that the frequency of the cleavage site of A is not so high. For that purpose, a type II restriction enzyme (rare cutter) that specifically recognizes a DNA base sequence longer than 6 bases and specifically cuts is required. .

[0003]

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

[0004]

Means for Solving the Problems The present invention will be summarized. The present invention relates to a restriction enzyme, which is represented by the following formula (Formula 1) in a double-stranded deoxyribonucleic acid:

[0005]

[Chemical 1]

(Where A is adenine, G is guanine, T
Is a thymine and C represents cytosine), and has the ability to specifically recognize the base sequence represented by thymine and C to specifically cleave it at the position of the arrow , and the following physicochemical properties : Streptomyces sp. AH1825 strain
(FERM BP-4836) or its mutant strain
It relates to a restriction enzyme that can . (A) Optimum temperature: about 37 ° C. (b) Optimum pH: pH 7.0 to 9.0 The second invention of the present invention relates to a genetic engineering reagent,
It is characterized by containing the restriction enzyme of the first invention. Further, a third invention of the present invention relates to a method for producing the restriction enzyme of the first invention, which is Streptomyces sp. AH18.
25 strain (FERM BP-4836) or its mutant strain is cultured, and the restriction enzyme is collected from the culture. The fourth invention of the present invention relates to the restriction enzyme-producing bacterium of the first invention, which produces the restriction enzyme of the first invention.
Streptomyces S., characterized by have the ability
P AH1825 strain (FERM BP-4836)
Relates to the mutant strain.

The type II restriction enzyme of the present invention may be any enzyme capable of specifically recognizing and cleaving the above-mentioned base sequence, and these enzymes can recognize the seven bases of the above double-stranded DNA. A recombinant strain having enzyme-producing ability, or a mutant strain of these strains, or a gene encoding the present enzyme-producing ability is isolated from these strains by using a normal gene manipulation method and introduced into another organism. Can be produced by any of the following.

Specific examples of the above-mentioned strain having the ability to produce a 7-base recognition restriction enzyme include Streptomyces sp. AH1825. The present bacterium is a strain newly obtained by the present inventors from soil, and its mycological properties are shown in Table 1 below.

[0009]

[Table 1] 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) the presence of aerial hyphae ^* 2 + Presence or absence of spore linkage ^{* 2} ＋ ───────────────────────────────────

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

The analysis of cell wall components is carried out by the following three publications,
Japan Society for Actinomycetes Study: “Experimental method for identifying actinomycetes”, (1st
(1985) Published by Japan Actinomycetes Society Secretariat, edited by Kazuo Komagata: "Methods for chemical classification of microorganisms", (first edition) (198)
2) Published by Academic Publishing Center and co-authored by Eiko Yabuuchi and others:
"Bacterial identification method that accompanies new taxonomy", (1st edition)
(1987) Nane Shuppan, and the cell wall type of this strain is type I. The quinone-based analysis has menaquinone having 6 or 8 saturated hydrogen atoms in the multiprenyl side chain and 9 isoprene units in the isoprenoid side chain. Further, the presence of aerial hyphae and spore linkage were observed by morphological observation. From the above results, this strain is identified as an actinomycete belonging to the genus Streptomyces.

This strain is Streptomyces s
p. It is named and displayed as AH1825, and has been deposited as FERM BP-4836 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

Streptomyces sp. AH182
The 7-base recognition restriction enzyme produced by 5 strains has the above-mentioned enzymatic action and is named Sse 1825I.

Restriction enzyme Sse 1825I according to the invention
The manufacturing method will be described in more detail. First, when culturing, the nutrient source added to the medium is used by the strain to be used, and Ss
As long as it produces e 1825I, glucose, maltose, glycerin and the like can be used as the carbon source, and yeast extract, peptone, corn steep liquor, meat extract and the like are suitable as the nitrogen source. In addition, inorganic salts such as phosphates, potassium salts, magnesium salts and metal salts may be added. Sse 1
Although the production amount of 825I varies depending on the culture conditions, generally, the culture temperature is 20 to 35 ° C., and the pH of the medium is 6 to 8.
Restriction enzyme Sse 182 in aerated stirred culture for ~ 3 days
The production of 5I reaches the maximum. The culture conditions are the strain used,
It is natural to set the maximum production amount according to the medium composition and the like. The restriction enzyme Sse 1825I produced by culturing the strain of the present invention is mainly present in the cells. The cells can be separated from the culture solution by centrifugation, for example. This enzyme can be extracted and purified by a method according to a general restriction enzyme purification method. For example, after suspending the bacterial cells in a buffer solution, the cells are disrupted by ultrasonication to extract the intracellular enzyme. Next, after removing cell debris by ultracentrifugation, the extract is salted out with ammonium sulfate. The precipitate was added to buffer A (10 mM potassium phosphate, pH 7.
5, 10 mM 2-mercaptoethanol, 5% glycerin), dialyzed against the same buffer, and purified by ion exchange chromatography, molecular sieve chromatography, affinity chromatography, etc. to obtain the restriction enzyme. it can.

The method for measuring the activity of the restriction enzyme Sse 1825I is shown below. 48 μl of a reaction solution having the composition shown in Table 2 below is preheated at 37 ° C. in advance, and then 2 μl of the present enzyme is added to bring the total amount to 50 μl, and the enzyme reaction is allowed to proceed. After 10 minutes, 5 μl of an enzyme reaction stop solution (1% SDS, 50% glycerin, 0.02% bromphenol blue) was added to stop the reaction.

[0016]

[Table 2] Table 2 ─────────────────────────────────── 10 mM Tris-HCl pH 8.0 10 mM MgCl ₂ 7 mM 2-mercaptoethanol 100 mM KCl 1.0 μg λ-DNA ────────────────────────────────────

The reaction solution is layered on a 0.7% agarose slab gel and electrophoresed under a constant voltage of 10 V / cm for about 1 to 2 hours. Electrophoresis buffer is 90 mM Tris-borate buffer (pH 8.3) 2.5 mM EDT
A is used. By including 0.5 μg / ml of ethidium bromide in the gel in advance, a DNA band can be detected by UV irradiation. The end point is when the number and amount of bands of the DNA fragment do not change.
The activity is defined as 1 unit of enzyme activity that completely cleaves 1 μg of λ-DNA at 37 ° C. for 1 hour.

The restriction enzyme Sse 1825I has the following physicochemical properties.

(1) Action and Substrate Specificity The present enzyme is an enzyme which recognizes the base sequence represented by the formula (Formula 1) in the double-stranded DNA sequence and cleaves at the position of the arrow. The recognition site of the restriction enzyme Sse 1825I of the present invention was determined as follows. Restriction enzyme Sse 1825
For I, λ-DNA was cut at 1 site and AD-2 DNA was cut at 8 sites. However, pUC18DNA, M13mp18D
NA, SV40 DNA, Col EI DNA, pBR
The 322 DNA and φX174 DNA were not cleaved. As a result of this, and 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]

[Chemical 2]

It was suggested that they were recognizing This 7
The base sequence contains a 5-base recognition restriction enzyme AvaII inside.
[Formula (Formula 3)]:

[0022]

[Chemical 3]

The inclusion of λ-DNA, A
Cleavage of each D-2 DNA with AvaII followed by Ss
Digested with e 1825I showed no change in the pattern of the obtained DNA fragment, and the restriction enzyme Sse 182
It was concluded that 5I recognizes formula (Formula 2).

Deoxyoligonucleotide having a recognition sequence for the restriction enzyme Sse 1825I for determining the cleavage site

[0025]

Embedded image 5′-AATTCCTCGAGAAGGGAC
CCAACCATGGA-3 '

[0026]

Embedded image 5′-GGAGCTCTTCCCTGGGGTT
GGTACCTTCGA-3 '

Was synthesized with a DNA synthesizer, annealed both, and then inserted into pUC118 and pUC119 for use. Single-stranded DNAs were prepared from both cleavage-determining plasmids by a conventional method. Primers that bind to adjacent sites of the multiple cloning site:

[0028]

[Chemical 6] 5'-GTTTTCCCAGTCACGAC-3 '

Fluorescently labeled 5'-end was prepared. The above-mentioned primers were annealed with single-stranded DNA prepared from both cleavage-determining plasmids, and then Bacillus caldotenax DNA was obtained.
Polymerase (BcaBEST DNA polymerase, Takara Shuzo)
Was used to synthesize a double-stranded DNA, the 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 7):

[0030]

[Chemical 7]

Detected as a band cleaved at the arrow mark of T4 DNA polymerase (T4 DNA Poly
merase, manufactured by Takara Shuzo Co., Ltd.), it was detected as a 3 bp long band by blunting treatment, and it was concluded that this enzyme recognizes the base sequence represented by the formula (Formula 1) and cleaves at the position of the arrow. Was done.

(2) Optimal enzyme activity condition I) Optimal temperature The optimal temperature of Sse 1825I was about 37 ° C. II) Optimum pH Sse 1825I has an optimum pH of 7.0 to 9.0.
Is in the range. III) Salt concentration The optimum salt concentration of Sse 1825I is 0 in the case of KCl.
Was ~ 150 mM. IV) MgCl ₂ concentration Sse 1825I has a MgCl ₂ concentration of 5 mM to ₂₀ m.
The enzymatic reaction was activated in the presence of M.

[0033]

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

Example 1 A medium 100 shown in Table 3 below was placed in a 50 ml Erlenmeyer flask.
The medium was charged and the medium was sterilized by a conventional method. Streptomyces sp. AH1825 (FERM BP-4836) cultured by shaking in the same medium as above at 30 ° C for 48 hours.
From 100 ml of the culture broth, 3.5 g of cells were obtained by cooling and centrifugation.

[0035]

[Table 3]                           Table 3       Glucose 10g       Yeast extract 10g       Polypeptone 10g       5g of salt       1 liter of deionized water                                           pH 7.2

The obtained 3.5 g of cells was added to 10 ml of buffer A (20 mM Tris-HCl, pH 7.5, 10 m).
M2-mercaptoethanol), and the mixture was crushed using an ultrasonic crusher and then centrifuged at 100,000 × G for 1 hour to remove the residue and obtain 12 ml of an extract. Ammonium sulfate was added to the resulting supernatant to 80% saturation, the precipitate was collected by centrifugation, dissolved in buffer A, and dialyzed overnight with the same buffer. Next, the dialysis solution was equilibrated with buffer solution A in advance, and phosphocellulose P11
It was adsorbed on a 5 ml column (manufactured by Whatman), washed with buffer solution A, and then eluted with buffer solution A having a linear concentration gradient of 0 to 1.0 M KCl. The obtained active fractions were combined and dialyzed with buffer A for 4 hours, and the dialyzed solution was adsorbed on a column of 2 ml of heparin-Sepharose (Pharmacia) equilibrated with buffer A in advance and washed thoroughly with buffer A. Then, it was eluted with buffer A having a linear concentration gradient of KCl of 0 to 1.0 M to obtain a preparation of this enzyme. This enzyme preparation was not contaminated with nonspecific DNA degrading enzyme and phosphatase. By the method described above, 600 units of activity was obtained from 3.5 g of wet cells.

[0037]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a restriction enzyme that recognizes and cleaves the 7-base sequence of double-stranded DNA. INDUSTRIAL APPLICABILITY The enzyme of the present invention is very useful in the field of genetic engineering for analysis of long chain DNA and the like.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 465) (72) Inventor Ikuno Kato 3-4-1 Seta 3-chome, Otsu City, Shiga Prefecture Central Research Institute In-house (56) References Gene, 1991, Vol. 109, No. 1, pages 121-3 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 9/14-9/46 JSTPlus (JOIS) BIOSIS / WPI (DIALOG) CA (STN)

Claims (4)

(57) [Claims] 1. The following formula (Formula 1) in the double-stranded deoxyribonucleic acid: (In the formula, A is adenine, G is guanine, T is thymine, and C is
Represents the cytosine) , and the ability to specifically recognize the base sequence represented by
And having the following physicochemical properties fine following list
Leptomyces sp. AH1825 strain (FERM
A restriction enzyme obtainable from BP-4836) or a mutant strain thereof . (A) Optimum temperature: about 37 ° C. (b) Optimum pH: pH 7.0 to 9.0 2. A reagent for genetic engineering, comprising the restriction enzyme according to claim 1. 3. Streptomyces sp. AH18
A method for producing a restriction enzyme, which comprises culturing 25 strains (FERM BP-4836) or a mutant strain thereof, and collecting the restriction enzyme according to claim 1 from the culture. 4. The ability to produce the restriction enzyme according to claim 1.
Streptomyces espi characterized by having power
-AH1825 strain (FERM BP-4836) or
That mutant strain.