JPH0330679A - Dna for coding ban i restriction endonuclease, ban i methylase and dna for coding ban i methylase - Google Patents

Abstract

NEW MATERIAL:A DNA fragment coding Ban I restriction endonuclease derived from Bacillus aneurinolytieus. EXAMPLE:DNA having a basic sequence coding an amino acid sequence expressed by the formula. USE:Collection of Ban I restriction endonuclease. PREPARATION:A chromosomal DNA collected from Ban I restriction endonuclease producing bacterium is digested with a proper restriction enzyme and integrated into a plasmid of BR 322, etc., and transformed to Escherichia coli and the transformed bacterium is proliferated to isolate Ban I restriction endonuclease producing bacterium. The Ban I restriction endonuclease is collected from culture of the producing bacterium and DNA coding the enzyme is isolated and identified.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides DNA encoding Ban I restriction endonuclease, Ban I methylase, and Ban I restriction endonuclease.
Concerning DNA encoding l methylase.

[Conventional technology]

So far, about 120 types of type 2 restriction enzymes have been discovered and commercialized from bacteria and other sources. Type 2 restriction enzymes are highly specific enzymes that recognize and cleave specific base sequences in deoxyribonucleic acid (DNA) chains, and are widely used in the field of genetic engineering. It is also expected to be used in clinical tests based on genetic diagnosis.

Ban I restriction endonuclease is one of the ■ type restriction enzymes. This enzyme is GGPyP in the DNA base sequence.
It is an enzyme that recognizes and cleaves uCC, and Bacillus
Bacillus aneur
This enzyme is produced in Bacillus aneurinolyticus IAM1077.
In addition to Ban 1, IAM1077 simultaneously produces other restriction enzymes Ban U and Ban III within its bacterial cells, making it extremely difficult to remove these enzymes, posing a manufacturing problem.

On the other hand, restriction site methylases methylate the nucleotide sequences recognized by restriction enzymes, and are used in genetic engineering to prevent cleavage by the corresponding restriction enzymes by methylating the recognition sequences of restriction enzymes. However, Ban I methylase, which methylates the base sequence recognized by Ban I restriction endonuclease, and its DNA
To date, no examples have been found where this has been obtained.

[Problems to be Solved by the Invention] Using genetic engineering technology, Bacillus aneurinolyticus
If it is possible to produce Ban I restriction endonuclease in a host other than the Bacillus aneurinolyticus
This solves the problem of contaminant enzymes derived from A. ticus and makes it possible to obtain pure an I restriction endonuclease. The problem of the present invention is to use this genetic engineering technology to
When producing Ban I restriction endonuclease, the first step is to provide the necessary DNA encoding the enzyme, and when the host retains the DNA encoding Ban I restriction endonuclease, the host's original In order to prevent DNA from being cleaved by Ban I restriction endonuclease, which inhibits host growth, it is necessary to provide the host with the ability to produce Ban I methylase, and Ban I methylase is essential for this purpose. The objective is to provide DNA encoding the

In addition, the object of the present invention also includes providing an I methylase itself, which is useful in various genetic engineering techniques.

[Means to solve the problem]

The present inventors collected chromosomal DNA from Ban I restriction endonuclease producing bacteria, digested the obtained chromosomal DNA with an appropriate restriction enzyme, integrated it into a plasmid such as pBR322, transformed Escherichia coli etc. with this plasmid, and transformed it. Grow the converted bacteria and use Ban ■ restriction endonuclease, or Ban! By isolating methylase producing bacteria and culturing these producing bacteria, Ban I
In addition to collecting restriction endonuclease and Ban I methylase, they also succeeded in isolating and identifying the DNA encoding these enzymes, leading to the completion of the present invention.

That is, the present invention provides: (1) Bacillus anoirinolyticus (Bacillus
Ban derived from lusaneurinolyticus)
DNA fragment encoding I restriction endonuclease.

(2) The DNA fragment according to (1), which has a base sequence encoding the amino acid sequence represented by the following formula I.

Formula 1 % Formula % (3) The DNA fragment according to (2), which has a base sequence represented by the following formula (■).

Formula■ GTTCCCACCC AATCTGTTTC TAAAGCATCT TGTTGTATTA TTACATAACA AAAGTGTTAG GGAAAATTTGA ATTTTTTGG Glu Leu Asn Val Leu Le
u Lys Phe lie LeuAAGGAGC
GTT ATATTTTTTGT ΔGCAGAA
TA ACAGGAGGGGGCTGAAGCAAA
TACGTTAGGT C to GTCGCTCAA
ACCCATGTGATATAAA 8^A GTAATGGAC8CAT GAAGGT GTGACCCATA AAGGCAGC GTAACTACA ^he AGCGTTG CGATTGAGAT T
AGCTTCCAGATAGTACCAT CGAG
CGT 888 GC hehe ACAAG TATAGCTT
AT to AAAATGCA^TTATCGATG TCACAACCAT GAGCGGGGAA CTTCCAACGTTTG
TAATAAT AGCCATTGC8ty 8rg G to u Lys Lys 1e Leu Tyr Arg 11e Gly Ala Ala Cys Val Ala Cys Glu Glu Thr Ser 8la Gly Ala Gly Val L eu Gly Ile Arg Lys Arg Phe Leu Ser 5er Glu Thr Tyr Pro Gln Gly Phe Pro Glu Phe Pro Cys Lys Gln Gln 8rg Asp Asn 11e Gly Phe Glu Glu Leu Glu ThrGlu li
e Asp Lys Ala Leu Asn PheAsp Ile
H4s Glu Phe Asp Phe Leu Gln Pro Phe 5erGly Phe
Gly Asp Arg Pro Lys Ala (4) Bacillus anoirinolyticus (Bacillus
Ban derived from usane'urinolyticus)
DNA fragment encoding l-methylase.

(5) The DNA fragment according to (4), which has a base sequence encoding an amino acid sequence represented by the following formula III.

2■ Met Lys Ile Lys Phe V
al Asp Leu Phe Ala1e Tyr 1e Leu Gly lie Leu Gly Ser Lys Ser Glu Lys Tyr Arg Cys S
er Asp Glu PhePhe Gin Asn
Asn Val Asn Glu Leu Phe
GlyAsn Phe Glu Leu Leu H4
s Gly Tyr Arg Leulle Asp
Tyr Arg Gly Gly Asn
Ser Ile Old 5Ser Trp Glu
Leu Gly rle Lys Gly Asp
Cys (6) Make the base sequence represented by the next 2■ (
5) The described DNA fragment.

2■ ATGAAGATTA AATTTGTTGA C
TTGTTTGCT GGAATTGGAGGAAT
TCGAAT AGGATTTGAA AGAGC
GGCCA AACGGTTTGA8AGGCTTG
To C ^ to ACATATGCCCTTAATTTT
GAGGAAC to A ys Val Val ilu Lys Val lie Glu Asp Leu 1;
TGGAGCAGCCGATT CTCACAAGTA TAAGAATGAA TGTATGA CAAATATCTT TATTTGATGA AAGCTATGCT (force (4) to (6) Expression vector containing the DNA fragment described in any one of (8) (7) A transformant transformed with the expression vector described in (7).

(9) The transformant according to (8), which is obtained by transforming a microorganism belonging to the genus Esherichia.

GO) Transform biological cells or microorganisms with the expression vector incorporating the DNA fragment described in any of (4) to (6), culture the obtained transformant, and collect Ban I methylase from the culture. A method for producing Ban I methylase, characterized in that:

(11) Bacillus anoirinolyticus
lusaneurinolyticus).
Ban I methylase with a molecular weight of 4000 (S[by S-PAGE method).

A Ban I methylase according to (11), which is a polypeptide having an amino acid sequence represented by the following formula III.

Formula■ Met Lys lie Lys Phe
Val Asp Leu Phe AlaGl
y Ile Gly Gly Ile Arg
Ile Gly Phe GluArg Ala
Ala Glu Cys Val Lys Ala Cys Lys Glu Glu 11e Thr 5er Leu Ala Gly Tyr Ala Gly Lys Leu 1u Pr.

Phe Phe Lys Arg Phe Ser 5er Thr Tyr Gin Gly Pro Glu Pro Cys Gin Gln Glu Leu Glu Glu Ile Asp Ala Leu Asn Asp Ile His Phe A sp Phe Gln Pro Phe Gly Phe Gly Thr Lys Phe Glu 0 Leu Ser ^5p 11e Gly Gln Leu Ser Lys V
al Vat Gly Asnrne Llin
Asn Asn Vat Asn Glu
Leu Phe GlyGin Met L
ys Leu Ala Asn Vat V
Regarding al.

The present invention will be explained in detail below.

Plasmids containing the Ban I restriction endonuclease gene of the invention are, for example, Escherichia
ia) Plasmids that take a form that can proliferate in cultured cells, such as the colicin El factor, which is known as an extrachromosomal gene (plasmid) of microorganisms belonging to the genus Bacillus aneurinolyticus.
lyticus) IAM1077-derived Ban I restriction endonuclease gene, a DNA fragment containing the Ban I methylase gene, and a DNA fragment containing only Ban I methylase, and the vector DNA is extracted from naturally occurring vector DNA. In addition to those that have been deleted, some DNA parts other than those essential for proliferation may be deleted (Co1E 1 strain,
Examples include pBR322 strain, psclo strain, 9M89 strain, and lambda phage strain.

Furthermore, any known method can be applied to incorporate the chromosomal DNA fragment into the vector DNA. For example, a method is used in which chromosomal DNA is cut at a specific site by treatment with an appropriate restriction enzyme, and then religated with similarly treated vector DNA using mixed gauze.

The pBR322 plasmid was used as the vector DNA, and Bacillus aneurynolyticus was injected into it.
aneurinolyticus) by incorporating the chromosomal DNA fragment prepared from IAM1077.
A new plasmid pBANIRM8 is obtained. pBA
The restriction enzyme map of the NIRM 8 plasmid is shown in FIG. As shown in Figure 1, this plasmid contains Bacillus neurinolyticus at the restriction site of the pBR322 plasmid.
DN containing the Ban I restriction endonuclease gene and Ban I methylase gene of IAM1077
This is a plasmid with an 8 kb base pair into which the A fragment has been integrated.

The gene encoding the Ban I restriction endonuclease and the Ban I methylase gene of the present invention can be identified as follows.

First, we used this plasmid strain Escherichia coli HB to identify the Ban I restriction endonuclease gene.
IOI (pBanlRM8) (FERM P-942
4) (Escherichia coli
colt) flea IBIOI (pBANll). ] was cultured to obtain bacterial cells.

Ban l restriction endonuclease, Ba
Electrophoresis (SO5PAGE) of each n I methylase
) to obtain a single band, and the N-terminal amino acid sequence of this sample was determined using an amino acid sequencer.

Furthermore, the above-mentioned strain producing Ban[restriction endonuclease was cultured, and a plasmid was prepared from the cultured cells.

This plasmid naturally contains DNA encoding Ban I restriction endonuclease and DNA encoding Ban I methylase, and can be used to determine the base sequence. To determine the DNA base sequence, a DNA fragment of the region thought to encode the above two enzymes from this plasmid was inserted into a vector suitable for base sequence determination, such as pUc18. Transfer to M13mplB, M13
-Dioxy method (Sanger, Pro, Natl
, Acad, Sci, 74.5463-54
67 (1977). That is, pUc18.
After changing the recombinant plasmid into M13mplB, various deletion mutants were created by determining the restriction enzyme site with various restriction enzymes, subcloning, deletion method using mung bean nuclease, and exonuclease. The region of the I methylase gene was determined. In addition, the inserted DNA of deletion mutants
The base sequence of the fragment was determined. Furthermore, in deletion mutants that express only the former of restriction enzyme activity and methylase activity, the bacteria die, and the DNA in the region where the base sequence should be determined cannot be obtained, so the base sequence may not be determined. In such cases, determination was made using a custom primer method or the like. As a result, the DNA sequence encoding the Ban I restriction endonuclease represented by the formula (1) and the DNA sequence encoding the Ban I methylase represented by the formula (2) were determined.

The N-terminal amino acid sequence determined from the base sequence is purified Ba
n I restriction endonuclease, Ban [matched the N-terminal amino acid sequence obtained from the methylase preparation. Using the DNA sequence determined here, each gene can be collected, and at the same time, using genetic manipulation techniques, Ban 1 restriction endonuclease, Ban
A production system for I methylase can be constructed. To produce substances using genetic engineering techniques, any system using Escherichia coli, yeast, or animal cells may be used.

(Japan Biochemistry Conference “Gene Research Methods If” pp126-
150. ap170-204 (1986)), for example, to produce Ban 1 restriction endonuclease using E. coli as a host, control sites for expression, such as the promoter of the tryptophan operon and the SD
The DNA sequence to which the sequence has been added may be ligated to a vector DNA such as pBR322 to which the Ban I methylase gene has been added, and then introduced into E. coli.

When yeast is used as a host, expression control sites include the inhibitory acid phosphatase gene and glyceraldehyde 3.
-The promoter of the phosphate dehydrogenase gene is known, and the origin of replication in yeast is 2 μm.
It is preferable to use those derived from plasmids or yeast chromosomes. When using animal cells as hosts, SV as the expression control site
40 promoter and IIB virus gene promoter are known, and it is preferable to use SV40 or polyoma virus promoters as replication starters. Also, Ran
If only the DNA encoding the Ran I restriction endonuclease of the present invention is used, the host may not be able to proliferate. If such an expression system is constructed, Ban I
Restriction endonucleases can be produced in large quantities. When it is desired to produce only Ban I methylase, only this enzyme can be produced in large quantities by using only the Ban I methylase gene in the above expression system.

The present invention will be specifically explained below using Examples.

Example 1 (1) Preparation of chromosomal DNA carrying the BanI restriction endonuclease gene
neurinolyticus) IAM1077 L
-broth medium (tryptone (Dirc) per 12 parts of pure water
o) 10g, yeast extract 5g, p NaCl Log
(Prepared to H7.0) Inoculated into 50ml and heated at 30°
A shaking culture was performed at C, and the bacterial cells were collected after 14 hours.

Next, the collected bacterial cells were mixed with lO■/- lysozyme (Taiyo Kagaku■
), 50m containing 20% sucrose, lmM EDT^
M) Suspended in 20 mQ of Lis-HCl buffer (p) (7,6) and allowed to stand at 37°C for 10 minutes. Next, a 0.1 M EDTA solution containing 1% lauroyl sarcosinate (pH 9) was added.
, 6) Pronase solution of 44 d and 5.44 d/d 2.
Od was added and left at 500°C for 30 minutes. Next, 66g of cesium chloride.

After adding 3.3 d of ethidium bromide solution of 10. Centrifugation was performed at OOOrpm for 40 hours. DNA was extracted with a syringe, ethidium bromide was removed by n-butanol extraction, and
Approximately 500 μg of chromosomal DNA was obtained by dialysis against IOIIM Tris-HCl buffer (pH 8, O) containing 1 mM EDTA.

(2) Insertion of chromosomal DNA fragment into vector Add 0.02 units of 5Au3AI restriction endonuclease to 3 μg of chromosomal DNA obtained in (1), and incubate at 37°C.
This was partially decomposed by carrying out a reaction for 11 hours. Next, vector plasmid pBR322 (Toyobo type)
By adding 4 units of BamHI restriction enzyme per 1 μg and carrying out the reaction at 37°C for 11 hours, it was completely degraded. Furthermore, by adding 1 unit of alkaline phosphatase and carrying out the reaction at 37°C for 11 hours,
The phosphoric acid at the end was removed.

Mix 3 μg of chromosomal DNA fragment obtained by the above method and 1 μg of pBR322 DNA fragment, and add 1 m
Plasmid DNA incorporating chromosomal DNA was obtained by performing a ligation reaction at 15°C for 116 hours using 5 units of T4 phage-derived DNA ligase in the presence of MATP and 5IIIM dithiothreitol.

(3) Transformation with a recombinant plasmid containing the BanI restriction endonuclease gene Escherichia 3988101, which is a hybrid strain of Escherichia coli 112 and Escherichia 398.
The strain was grown in LB medium (11 parts tryptone (Dirco) in pure water).
10g, yeast extract 5g, NaC 110g @ pH 7
, 0) was inoculated into lOId, cultured with shaking at 37°C, grown to logarithmic growth phase, and then harvested. This was cooled with water to a final concentration of 0.03M CaC1z.
The cells were suspended in a solution of The plasmid DNA solution obtained in (2) was added to this cell suspension, reacted for 60 minutes under water cooling, and heat shocked at 42°C for 1-2 minutes to incorporate the plasmid DNA into the cells. Next, this cell suspension was separately inoculated into the above LB medium, cultured with shaking at 37'C for 35 hours, and isolated from Ban [A strain producing restriction endonuclease, N. coli)IBIOI ( pBan
lRM8) (FERM P-9424) was obtained. Plasmid pBanlRM8 contained in this transformed microorganism
The restriction enzyme map of is shown in Figure 1. Also pBanIRM8
Escherichia coli (pBanIM 4) was subcloned to isolate a strain that produces Ban I methylase.
) (FERM P-10741) was obtained.

(4): L'y Erichia coli HBOI (pB
Production of Banl restriction endonuclease by anlllM8) (3)
RM P-9424) was cultured with aeration and stirring at 37°C for 16 hours in a 200 L fermentation tank using the LB medium. This was centrifuged to collect bacteria, yielding 385 g of cultured bacteria. Ban I restriction endonuclease was purified from 100 g of the obtained bacterial cells. 100g of bacterial cells was added to BufferA (10mM
Mgch was suspended in 20mM Tris-HCl buffer pH 7.5) containing 7mM 2-mercaptoethanol. This suspension was crushed for 10 minutes using an ultrasonic crusher, a sonicator, to prepare a supernatant. Supernatant liquid to 0゜2M
Polyethyleneimine was added at a concentration of 0.2% in the presence of NaCl, and centrifugation was performed at 8000 rpm for 20 minutes to obtain a supernatant. To this supernatant, ammonium sulfate was added to saturation of 70%, and after ammonium sulfate precipitation, the washed product was collected, and 10 mM MgC1z, 7
The suspension was suspended in 20 mM Tris-HCl buffer (pH 7.5) containing mM 2-mercaptoethanol and dialyzed. After dialysis, phosphocellulose force ram (volume 200 d) chromatography was performed. Ran 1 restriction endonuclease was eluted at a KCI concentration of around 0.5M. The eluted enzyme solution was placed in a dialysis tube and dialyzed. The dialysate was further subjected to hydroxyapatite column (80In1) chromatography. The Ban I fraction was eluted at a phosphoric acid concentration of 0.3M. Furthermore, when this fraction was dialyzed and subjected to DEAE-Sepharose (40d) chromatography, Ran
It was observed that I was eluted in the wash fraction. Both fractions were concentrated and then subjected to Sephacryl S-200 gel filtration. Put the gel filtration Ban I fraction into a dialysis tube and add 2a+M
An enzyme solution of 2.8 In1 was obtained by dialysis against a phosphate buffer containing mercaptoethanol and 50% glycerol.

When this purified Ban I enzyme was examined for protein purity using the 5DS-PAGE method, it was found to be a single 13an I protein. Furthermore, the molecular weight of Ban I was approximately 72.000 (SO5-PAG) due to the elution position of Sephacryl 5-200.
It was estimated to be a dimer of 136,000 subunit molecules (Fig. 2). When the N-terminal amino acid sequence of this enzyme preparation was determined using an amino acid sequencer, AlaG
ln-Leu-Lys-Tyr-Asn-Lys-A
It turned out to be sp-I 1e-Asp-G lu-LeuGlu.

(5) Transformation with a recombinant plasmid containing the BanI methylase gene From the recombinant plasmid pBanlRM8 containing Banl restriction endonuclease DNA, the BanIII-5ph I fragment containing only the methylase gene was transformed into pUc.
Insert into the Accf-sph I site of 18 pBanl
M4 was prepared (Figure 3). Then pBanlM4
E. coli HBOI was transformed using the conventional method.

(6) Escherichia coli HBOI (pBan I
Production of Ran I methylase by pBa containing DNA encoding Ban I methylase
E. coli HBOI carrying nlM4 (pB
anlM4) (FIERM P-10741) to L-b
The cells were cultured in Roth medium using a 0.52 flask and the cells were collected. The bacterial cells were suspended in 50 ml of Buffer A and then disrupted by ultrasonication (3A, 10 minutes). 35,000rpm
, and centrifuged for 30 minutes to obtain a supernatant. This supernatant was subjected to phosphocellulose column (40d) chromatography. Since Ban I methylase passed through the column, it was collected and subjected to DEAE column chromatography. Ban I methylase is eluted with KCI 0.3M. Both parts are combined again with a phosphocellulose ram (2
0d) Chromatography was performed. Ban 1 methylase is 0
．． Eluted with 45M KCI. Both fractions were concentrated and subjected to Sephacryl S-200 gel filtration (2 cm x 37 cm). The Ban I methylase fraction was dialyzed and further subjected to heparin (10d) column chromatography. The eluted fraction was concentrated with polyethylene glycol and then dialyzed against a 50% glycerol solution. Total active 4 kilounits Ban
I methylase was obtained.

When the white purity of this specimen was examined by 5O3-PAGE and silver staining followed by injection staining, it showed a single band (Fig. 8). Also in this case, the molecular weight was about 44,000. In the gel filtration method, it was about 45,000 (Figure 4). This enzyme was estimated to be a monomer enzyme.

(7) Ban I restriction endonuclease, Ban
The strain Escherichia coli HBIOI (pBan
lRM 8 ) (FERM P-9424) was cultured and a recombinant plasmid (pBanlRM 8 ) was obtained by a conventional method.
was prepared. The prepared pBanllllMB was cut with restriction enzymes old ndlI[, 5phI, and DNA coated with Ban 1 restriction endonuclease and Ban I methylase was extracted.
Cut out the A fragment (6.2 kb) and convert it to Klenow fragment [
・After blunt-ending, this was converted into the old nclI of pUclB.
I inserted it into the site and created ρBanlRM6.2 (5th
figure). pBanlRM inserted into this ptlc18
A restriction enzyme map of the DNA fragment derived from No. 8 is shown (Fig. 6).
.

Furthermore, the coding regions of Ban I restriction endonuclease and Ban I methylase in this insert fragment are shown in FIG. In the figure, BanIR represents the coding region of Ban I restriction endonuclease;
IM represents the coding region of Ban I methylase. Note that Cmp 1 to 4 indicate custom mate primers used to determine the base sequence.

Next, pBanlRM6.2 was digested with restriction enzymes; BamHI,
Cut with Kpnl and convert pnl DNA into BamHI-
Fragments were prepared. This DNA fragment was subjected to gradual deletion from the Ban I restriction endonuclease gene side using exonuclease ■ and mung bean nuclease.
n), the resulting gradual deresicon mutants were created, and their sequences were determined using the dioxy method. This allows 1. The base sequence of the other strand was determined.

Regarding the other strand, nucleotides (17 mar) complementary to the base sequence determined as described above were synthesized, and the base sequence was determined using the dioxy method using these as primers. As a result, DNA encoding Ban I restriction endonuclease shown in the formulas ① and ① and Ban I
The base sequence of the DNA encoding methylase was determined.

In addition, Formula I and Formula ■ corresponding to the base sequences of ■ and ■
The amino acid sequence of (4) and (6) at the N-terminus is
), the N-terminal amino acid sequences of Ban I restriction endonuclease and Ban I methylase were respectively identical.

[Effects of the Invention] According to the present invention, it was possible to provide DNA encoding Ran 1 restriction endonuclease and DNA encoding Ban I methylase. This makes it possible to efficiently produce a pure an I restriction endonuclease preparation containing no contaminant enzymes in large quantities by using genetic engineering technology. Furthermore, the Ban[methylase provided by the present invention itself is extremely important as a reagent, etc. in genetic engineering technology, and will greatly contribute to the development of genetic engineering technology.

[Brief explanation of drawings]

Figure 1 is a restriction enzyme map of the pBanlRM 8 plasmid, Figure 2 is a diagram showing the measurement results of the molecular weight of pBan I restriction endonuclease, and Figure 3 is a diagram showing the results of measuring the molecular weight of pBan I restriction endonuclease.
8 to pBanlM 4, Figure 4 shows Ran
Figure 5 shows the measurement results of I-notylase molecular weight.
Construction diagram of BanlRM6.2, Figure 6 is pBanlRM
pBan inserted into pUclB when creating 6.2
Figure 7 shows the restriction enzyme map of the DNA fragment derived from pUclB.
FIG. 2 shows the coding regions of Ban 1 restriction endonuclease and Ban I methylase in the NA fragment and the strakigy of DNA sequencing of these enzymes. FIG. 8 is a photograph showing the electrophoretic pattern of Ban I methylase obtained according to the present invention.

Claims (1)

[Claims] 1. Bacillus aneurinolyticus
A DNA fragment encoding the BanI restriction endonuclease derived from A. neurinolyticus. 2. The DNA fragment according to claim 1, which has a base sequence encoding an amino acid sequence represented by the following formula I. Formula I
10 Met Ala Gln Leu Lys Tyr A
sn Lys Asp Ile

20 Asp Glu Leu Glu Arg Asn A
la Ala Lys Trp

30 Trp Pro Asp Phe Leu Ala L
ys Lys Glu Ser

40 Ser Thr Ser Ile Ile Pro L
ys Leu Val Glu

50 Ser Gln Asp Ala Phe Ile S
Er Leu Leu Asn

60 Leu Ser Lys Asn Asn Pro P
he Asp Ile Phe

70 Gln Leu Ile Asp Ala Ser L
ys Phe Pro Pro

80 Asn Leu Phe Leu Lys His L
eu Val Val Leu

90 Thr Asp Phe Gly Gly Glu P
ro Leu Asn Arg

100 Leu Asn Gln Asn Phe Asp S
Er Leu Phe Pro

110 Met Ile Pro Tyr Gly Ile H
is Tyr Ile Thr

120 Lys Val Leu Gly Lys Phe G
lu Phe Phe Trp

130 Asn Glu Lys Lys Tyr Glu T
yr Val Phe Gln

140 Glu Leu Pro Val Thr Ser L
eu Thr Asn Ser

150 Lys Leu Lys Ile Asp Gly A
la Ser Ile Ser

160 Lys Thr Val Pro Leu Ser A
sp Leu Tyr Lys

170 Asp Val Ile Val Leu Leu M
et Phe Gly Ala

180 Asn Ala Val Asn Ser Glu V
al Ser Glu Val

190 Leu Met Lys Cys Glu Val G
ly Asn Leu Ile

200 Gly Lys Thr Asp Glu Leu L
ys Lys Phe lle

210 Lys Glu Arg Tyr lle Phe V
al Ser Arg Ile

220 Thr Gly Gly Ala Glu Ala A
sn Thr Leu Gly

230 Gln Val Ala Gln Thr His V
al Ile Asp Phe

240 Leu Arg Thr Arg Phe Gly S
er Lys Gly His

250 Asp Ile Lys Ser Asn Gly H
is Ile Glu Gly

260 Val Thr His Asn Asp Gly G
ln Thr Leu Thr

270 Thr Phe Asp Val Val Ile L
ys Lys Gly Ser

280 Lys Ser Val Ala Ile Glu I
Ser Phe Gln

290 Val Thr Thr Asn Ser Thr I
le Glu Arg Lys

300 Ala Gly Gln Ala Lys Ala A
rg Tyr Asp Met

310 Val Ser Asp Thr Gly Asn T
yr Ile Ala Tyr

320 Ile Ile Asp Gly Ala Gly A
sn Phe Gln Arg

330 Lys Asn Ala Ile Thr Thr I
le Cys Asn Asn

340 Ser His Cys Thr Val Ala T
yr Tyr Glu Glu

350 Glu Leu Asn Val Leu Leu L
ys Phe Ile Leu Glu Lys Le
u Glu 3, the DNA fragment according to claim 2, having a base sequence represented by the following formula II. Formula II 10 20
30 ATGGCACAGC
TAAAATACAA TAAAGATATT GA
TGAAT 40 TGG 50 60
70 AAAGAAACGC
TGCAAGTGG TGGCCGGATT TT
CTAGC 80 TAA 90 100
110 AAAGGAGTCG
TCAACGAGCA TTATTCCTAA AC
TGGTC120 GAA 130 140
150 TCGCAAGATG
CTTTATTATATC
CACTA160 AGA 170 180
190 ACAACCCTTT
CGACATTTTC CAACTAATAG AC
GCATC200 TAA 210 220
230 GTTCCCACCC
AATCTGTTTC TAAAGCATCT TG
TTGTA240 TTA 250 260
270 ACCGACTTTG
GTGGAGAGCC ACTAAATCGC TT
GAATC280 AAA 290 300
310 ACTTTGATTC
GCTTTTTTCCC ATGATTCCCT AT
GGAAT320 CCA 330 340
350 TTACATAACA
AAAGTGTTAG GGAAATTGA AT
TTTTT360 TGG 370 380
390 AATGAGAAAA
AATATGAATA TGTTTTTTCAA GA
ACTTC400 CGG 410 420
430 TTACATCATT
AACTAATTCA AAACTCAAGA TT
GACGG440 ACC 450 460
470 AAGTATTTCT
AAGACAGTCC CACTTTCTGA TT
TATAT480 AAA 490 500
510 GATGTAATTG
TATTACTCAT GTTCGGTGCT AA
TGCTG520 TTA 530 540
550 ATTCGGAAGT
ATCCGAAGTT TTGATGAAAT GT
GAAGT560 TGG 570 580
590 AAATCTCATT
GGTAAGACTG ACGAGTTGAA GA
AGTTT600 ATT 610 620
630 AAGGAGCGTT
ATATTTTTTGT AAGCAGATA AC
AGGAG640 GGG 650 660
670 CTGAAGCAAA
TACGTTAGGT CAAGTCGCTC AA
ACCCA680 TGT 690 700
710 AATAGATTTT
CTAAGAACTC GATTCGGGTC TA
AGGGT720 CAC 730 740
750 GATATAAAAAA
GTAATGGACA CATTGAAGGT GT
GACCC760 ATA 770 780
790 ACGATGGACA
AACACTAACC ACTTTTGATG TA
GTCAT800 AAA 810 820
830 AAAAGGCAGC
AAAAGCGTTG CGATTGAGAT TA
GCTTC840 CAG 850 860
870 GTAACTACAA
ATAGTACCAT CGAGCGTAAA GC
TGGAC880 AAG 890 900
910 CTAAAGCACCG
ATACGATATGGTTAGTGATACT
GGCAA920 TTA 930 940
950 TATAGCTTAT
ATTATCGATGGAGCGGGGAACT
TCCAA960 CGT 970 980
990 1AAAAAATGCAA
TCACAACCAT TTGTAATAAT AG
CCATT000 GCA 1010 1020
1030 1CAGTAGCTTA
TACCGAAGAA GAACTAAATG TC
TTGCT040 AAA 1050 1060 ATTCATCTTA GAAAAAAACTTG AA 4, Bacillus aneurinolyticus
A DNA fragment encoding BanI methylase derived from A. neurinolyticus. 5. The DNA fragment according to claim 4, which has a base sequence encoding an amino acid sequence represented by the following formula III. Formula III
10 Met Lys Ile Lys Phe Val A
sp Leu Phe Ala

20 Gly Ile Gly Gly Ile Arg I
le Gly Phe Glu

30 Arg Ala Ala Lys Arg Phe G
lu Leu Glu Thr

40 Glu Cys Val Leu Ser Ser G
lu Ile Asp Lys

50 Lys Ala Cys Glu Thr Tyr A
la Leu Asn Phe

60 Lys Glu Glu Pro Gln Gly A
sp Ile His Glu

70 Ile Thr Ser Phe Pro Glu P
he Asp Phe Leu

80 Leu Ala Gly Phe Pro Cys G
ln Pro Phe Ser

90 Tyr Ala Gly Lys Gln Gln G
ly Phe Gly Asp

100 Thr Arg Gly Thr Leu Phe P
he Glu Val Glu

110 Arg Val Leu Arg Asp Asn A
rg Pro Lys Ala

120 Phe Leu Leu Glu Asn Val A
rg Gly Leu Val

130 Thr His Asp Lys Gly Arg T
hr Leu Lys Thr

140 Ile Ile Ser Lys Leu Glu G
lu Leu Gly Tyr

150 Gly Val Ser Tyr Leu Leu L
eu Asn Ser Ser

160 Thr Phe Gly Val Pro Gln A
sn Arg Val Arg

170 Ile Tyr Ile Leu Gly Ile L
eu Gly Ser Lys

180 Pro Lys Leu Thr Leu Thr S
er Asn Val Gly

190 Ala Ala Asp Ser His Lys T
yr Lys Asn Glu

200 Gln Ile Ser Leu Phe Asp G
lu Ser Tyr Ala

210 Thr Val Lys Asp Ile Leu G
lu Asp Ser Pro

220 Ser Glu Lys Tyr Arg Cys S
er Asp Glu Phe

230 Ile Gly Gln Leu Ser Lys V
al Val Gly Asn

240 Asn Phe Glu Leu His G
ly Tyr Arg Leu

250 Ile Asp Tyr Arg Gly Gly A
sn Ser Ile His

260 Ser Trp Glu Leu Gly Ile L
ys Gly Asp Cys

270 Thr Lys Glu Glu Ile Glu P
he Leu Asn Gln

280 Leu Ile Ala Asn Arg Arg L
ys Lys Ile Tyr

290 Gly Thr His Gln Asp Gly L
ys Ala Leu Thr

300 Leu Glu Gln Ile Arg Thr P
he Tyr Asn His

310 Asp Gln Leu Glu Val Ile I
Le Lys Ser Leu

320 Leu Gln Lys Gly Tyr Leu A
rg Glu Glu Glu

330 Asn Lys Phe Asn Pro Vat C
ys Gly Asn Met

340 Ser Phe Glu Val Phe Lys P
he Leu Asp Pro

350 Asp Ser Ile Ser Ile His L
eu Thr Ser Ser

360 Asp Ala His Lys Leu Gly V
al Val Gln Asn

370 Asn Val Pro Arg Arg Ile T
hr Pro Arg Glu

380 Cys Ala Arg Leu Gln Gly P
he Pro Asp Asp

390 Phe Ile Leu His Ser Asn A
sp Asn Phe Ala

400 Tyr Lys Gln Leu Gly Asn S
er Val Thr Val

410 Lys Val Val Glu Lys Val I
Le Glu Asp Leu

420 Phe Gln Asn Asn Val Asn G
lu Leu Phe Gly Gln Met Ly
s Leu Ala Asn Val Val 16, having a base sequence represented by the following formula IV.
The described DNA fragment. Formula IV
ATGAAGATTA
AATTTGTTGA CTTGTTTGCT GG
AATTG 40 GAG GAATTCGAAT AGGATTTGAA AGA
GCGGCCA AACGGTT 80 TGA ATTGGAGACT GAGTGTGTAC TTT
CTAGTGA GATCGAC120 AAA
AAGGCTTGCG
AAACATATGC CCTTAATTTT AA
AGAGG160 AAC CTCAAGGTGA TATACACGAA ATT
ACCAGCT TTCC AGA200 ATT CGATTTTCTT CTTGCTGGAT TTC
CTTGTCA ACCCTTT240 TCA TATGCAGGTA AACAACAAGG ATT
TGGGGAT ACAAGAG280 GAA CATTATTTTTT TGAGGTGGAG AGG
GTTCTTA GGGATAA320 CCG CCCAAAGGCT TTCTTATTAG AAA
ATGTTCG GGGACTG360 GTA ACACACGACA AAGGAAGAAC TTT
AAAGACG ATCATTT400 CAA AATTAGAGGA ATTGGGTTAC GGG
GTTAGCT ACCTATT440 ACT TAACAGTAGT ACGTTTGGTG TCC
CCCAAA CCGAGTA480 AGG ATTTATATT TAGGCATACT CGG
AAGTAAA CCAAAGT520 TAA CTCTAACCTC TAATGTTGGA GCA
GCCGATT CTCACAA560 GTA TAAGAATGAA CAAATATCTT TAT
TTGATGA AAGCTAT600 GCT ACTGTTAAAG ACATTTTAGA AGA
TTCTCCA AGTGAAA640 AAT ATCGCTGTTC TGACGAATTT ATC
GGTCAACTTTCAAA680 GGT
TGTTGGTAAT
AACTTTGAAT TGCTACATG CT
ATCGC720 TTA ATTGATTATC GTGGCGGTAA TTC
GATTCAT TCTTGGG760 AAC TTGGCATCAA GGGTGATTGT ACA
AAAGAGG AAATTGA800 ATT TCTAAATCAG TTGATTGCAA ATA
GAAGAAA GAAAAATT840 TAT GGTACGCACC AAGACGGAAA AGC
CTTAACT TTAGAGC880 AAA TTCGGACGTT TTATAACCAC GAT
CAGCTTG AGGTAAT920 AAT AAAATCCCCTA CTCCAAAAAG GAT
ATTTAAG AGAAGAA960 GAA
1AATAAAATTT
A ATCCCGTTTG TGGGAATATG T
CTTTTG000 AGG
1TCTTTAAGTT
CCTTGACCCA GACAGTATTT CA
ATCCA040 CCT


1CACATCTAGCGATGCTCAT
A AGTTGGGAGT AGTTCAG080 AAT
1AATGTTCCTA
GAAGGATAAC ACCTCGGGAA TG
TGCTA120 GAC
1TGCAAGGTT
CCCAGATGAT TTCATACTAC AT
TCCAA160 TGA
1CAATTTTTGCA
TACAAGCAGT TGGGGAATTC AG
TCACT200 GTC
1AAAGTAGTG
AAAAAAGTGAT CGACGATTTA TT
TCAAA240 ACA
1ATGTAAATGA
ATTGTTTGGA CAGATGAAAC TT
GCAAA280 TGT TGTATGA 7, an expression vector comprising the DNA fragment according to any one of claims 4 to 6. 8. A transformant transformed with the expression vector according to claim 7. 9. The transformant according to claim 8, which is obtained by transforming a microorganism belonging to the genus Esherichia. 10. Transform biological cells or microorganisms with the expression vector incorporating the DNA fragment according to any one of claims 4 to 6, culture the obtained transformant, and extract B from the culture.
Ban characterized by collecting an I methylase
I Method for producing methylase. 11. Bacillus aneurynolyticus (Bacillus
saneurinolyticus), approximately 4,40
B with a molecular weight of 00 (by SDS-PAGE method)
an I methylase. 12. The Ban I methylase according to claim 11, which is a polypeptide having an amino acid sequence represented by the following formula III. Formula III
10 Met Lys Ile Lys Phe Val A
sp Leu Phe Ala

20 Gly Ile Gly Gly Ile Arg I
le Gly Phe Glu

30 Arg Ala Ala Lys Arg Phe G
lu Leu Glu Thr

40 Glu Cys Val Leu Ser Ser G
lu Ile Asp Lys

50 Lys Ala Cys Glu Thr Tyr A
la Leu Asn Phe

60 Lys Glu Glu Pro Gln Gly A
sp Ile His Glu

70 Ile Thr Ser Phe Pro Glu P
he Asp Phe Leu

80 Leu Ala Gly Phe Pro Cys G
ln Pro Phe Ser

90 Tyr Ala Gly Lys Gln Gln G
ly Phe Gly Asp

100 Thr Arg Gly Thr Leu Phe P
he Glu Val Glu

110Arg Val Leu Arg Asp A
sn Arg Pro Lys Ala

120 Phe Leu Leu Glu Asn Val A
rg Gly Leu Val

130 Thr His Asp Lys Gly Arg T
hr Leu Lys Thr

140 Ile Ile Ser Lys Leu Glu G
lu Leu Gly Tyr

150 Gly Val Ser Tyr Leu Leu L
eu Asn Ser Ser

160 Thr Phe Gly Val Pro Gln A
sn Arg Val Arg

170 Ile Tyr Ile Leu Gly Ile L
eu Gly Ser Lys

180 Pro Lys Leu Thr Leu Thr S
er Asn Val Gly

190 Ala Ala Asp Ser His Lys T
yr Lys Asn Glu

200 Gln Ile Ser Leu Phe Asp G
lu Ser Tyr Ala

210 Thr Val Lys Asp Ile Leu G
lu Asp Ser Pro

220 Ser Glu Lys Tyr Arg Cys S
er Asp Glu Phe

230 Ile Gly Gln Leu Ser Lys V
al Val Gly Asn

240 Asn Phe Glu Leu His G
ly Tyr Arg Leu

250 Ile Asp Tyr Arg Gly Gly A
sn Ser Ile His

260 Ser Trp Glu Leu Gly lle L
ys Gly Asp Cys

270 Thr Lys Glu Glu Ile Glu P
he Leu Asn Gln

280 Leu Ile Ala Asn Arg Arg L
ys Lys Ile Tyr

290 Gly Thr His Gln Asp Gly L
ys Ala Leu Thr

300 Leu Glu Gln Ile Arg Thr P
he Tyr Asn His

310 Asp Gln Leu Glu Val Ile I
Le Lys Ser Leu

320 Leu Gln Lys Gly Tyr Leu A
rg Glu Glu Glu

330 Asn Lys Phe Asn Pro Val C
ys Gly Asn Met

340 Ser Phe Glu Val Phe Lys P
he Leu Asp Pro

350 Asp Ser Ile Ser Ile His L
eu Thr Ser Ser

360 Asp Ala His Lys Leu Gly V
al Val Gln Asn

370 Asn Val Pro Arg Arg Ile T
hr Pro Arg Glu

380 Cys Ala Arg Leu Gln Gly P
he Pro Asp Asp

390 Phe Ile Leu His Ser Asn A
sp Asn Phe Ala

400Tyr Lys Gln Leu Gly A
sn Ser Val Thr Val

410 Lys Val Val Glu Lys Val I
Le Glu Asp Leu

420 Phe Gln Asn Asn Val Asn G
lu Leu Phe Gly Gln Met Ly
s Leu Ala Asn Val Val