Method for cloning and expression of Bpml restriction endonuclease in E. coli

The present invention relates to recombinant DNA which encodes the BpmI restriction endonuclease as well as BpmI methyltransferase, expression of BpmI restriction endonuclease from E. coli cells containing the recombinant DNA. BpmI endonuclease is a fusion of two distinct elements with a possible structural domains of restriction-methylation-specificity (R-M-S). This domain organization is analogous to the type I restriction-modification system with three distinct subunits, restriction, methylation, and specificity (R, M, and S). Because BpmI is quite distinct to other type IIs restriction enzymes, it is proposed that BpmI belongs to a subgroup of type II restriction enzymes called type IIf (f stands for fusion of restriction-modification-specificity domains). The Type IIf group of restriction enzyme includes Eco57I, BpmI, GsuI, BseRI and some other restriction enzymes that cut downstream sequences at long distance, 10-20 bp downstream of recognition sequence, such as MmeI (N20/N18)).

1. Isolated DNA coding for the BpmI restriction endonuclease, wherein the isolated DNA is obtainable from Bacillus pumilus (New England Biolabs collection #711).

2. A recombinant DNA vector comprising a vector into which a DNA segment encoding the BpmIRM restriction endonuclease has been inserted.

3. Isolated DNA encoding the BpmI restriction endonuclease and BpmI methylase M1, wherein the isolated DNA is obtainable from ATCC No. PTA-2598.

4. A cloning vector which comprises the isolated DNA of claim 3.

5. A host cell transformed by the vector of claims 2 or 4.

6. A method of producing recombinant BpmI restriction endonuclease comprising culturing a host cell transformed with the vector of claims 2 or 4 under conditions suitable for expression of said endonuclease.

The present invention relates to recombinant DNA which encodes the BpmI restriction endonuclease as well as BpmI methyltransferase and expression of BpmI restriction endonuclease from E. coli cells containing the recombinant DNA. BpmI is an isoschizomer of GsuI (Fermentas 2000-2001 Catalog, Product No. ER0461/ER0462).

Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.

Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecule. Once bound, they cleave the molecule within, to one side of, or to both sides of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313 (1999)).

Restriction endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′TTT/AAA3′, 5′PuG/GNCCPy3′ and 5′CACNNN/GTG3′ respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5′G/AATTC3′.

A second component of bacterial restriction-modification (R-M) systems are the methyltransferases (methylases). These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5-methyl cytosine, N4-methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.

By means of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex genomic DNA libraries, i.e. populations of clones derived by ‘shotgun’ procedures, when they occur at frequencies as low as 10⁻³to 10⁻⁴. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.

A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719 (1980); HhaII: Mann et al., Gene 3:97-112 (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507 (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophage, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phages. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.

Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985); Tsp45I: Wayne et al. Gene 202:83-88 (1997)).

A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since R-M genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241 (1983)).

A more recent method, the “endo-blue method”, has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of E. coli containing the dinD: :lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535, (1996); Fomenkov et al., Nucl. Acids Res. 22:2399-2403 (1994)). This method utilizes the E. coli SOS response signals following DNA damages caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535).

Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for creating recombinant molecules in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such overexpression strains should also simplify the task of enzyme purification.

The present invention relates to a method for cloning the BpmI restriction endonuclease from Bacillus pumilus into E.coli by methylase selection and inverse PCR amplification of the adjacent DNA of the BpmI methylase gene.

The present invention relates to recombinant BpmI and methods for producing the same. BpmI restriction endonuclease is found in the strain of Bacillus pumilus (New England Biolabs' strain collection #711). It recognizes double-stranded DNA sequence 5′ CTGGAG 3′ (or 5′CTCCAG3′) and cleaves 16/14 bases downstream of its recognition sequence (N16/N14) to generate a 2-base 3′ overhanging ends.

By methylase selection, a methylase gene with high homology to amino-methyltransferases (N6-adenine methylases) was found in a DNA library. This gene was named BpmI M1 gene (BpmIM1, 1650 bp), encoding a 549-aa protein with predicted molecular mass of 63,702 daltons. There was one partial open reading frame upstream of BpmIM1 gene that displayed 31% amino acid sequence identity to another restriction enzyme Eco57I with similar recognition sequence (Eco57I recognition sequence: 5′CTGAAG N16/N14; BpmI recognition sequence: 5′ CTGGAG N16/N14; A. Janulaitis et al. Nucl. Acids Res. 20:6051-6056, (1992)).

In order to clone the rest of the BpmIRM gene, inverse PCR was used to amplify the adjacent DNA sequence. After four rounds of inverse PCR reactions, an open reading frame of 3030 bp was found upstream of BpmI M1 methylase gene, which encodes a 1009-aa protein with predicted molecular mass of 116,891 daltons. By amino acid sequence comparison of BpmI endonuclease with all known proteins in GenBank protein database, it was discovered that BpmI endonuclease is a fusion of two distinct elements with a possible structural domains of restriction-methylation-specificity (R-M-S). This domain organization is analogous to the type I restriction-modification system with three distinct subunits R, M, and S. Because BpmI is quite distinct to other type IIs restriction enzymes, it is proposed that BpmI belongs to a subgroup of type II restriction enzymes called type IIf (f stands for fusion of restriction-modification domains).

To generate a premodified expression host, the BpmIM1 gene was amplified in PCR and cloned in E. coli strain ER2566. BpmI M1 methylase also modifies XhoI site. XhoI recognition sequence 5′ CTCGAG 3′ is similar to BpmI recognition sequence 5′ CTGGAG 3′ with only one base difference. It was concluded that BpmI M1 methylase may recognize the sequence 5′ CTNNAG 3′ and possibly modify the adenine base to create N6-adenine in the symmetric sequence.

The expression of 3030-bp BpmIRM gene was quite difficult because of the large size of the PCR porduct. The BpmIRM gene was first amplified by Taq DNA polymerase and cloned into the premodified host, but no BpmI activity was detected. To improve the fidelity of PCR reaction, Deep Vent DNA polymerase was used in PCR. Among 18 clones with the insert, only one clone (#4) displayed partial BpmI activity. This clone was sequenced and confirmed to contain wild type sequence.

FIG. 1 Gene organization of BpmI restriction-modification system. Genes BpmIRM and BpmIM1 code for BpmI endonuclease (BpmI endonuclease-methylase fusion protein and BpmI M1, respectively. BpmI-Δ#1, BpmI-Δ#2, and BpmI-Δ#3 are deletion mutants with deletions in the methylation or specificity domains.

FIG. 2 DNA sequence of BpmI M1 methylase gene (BpmIM1) (SEQ ID NO:1) and its encoded amino acid sequence (SEQ ID NO:2).

FIG. 3 DNA sequence of BpmI endonuclease gene (BpmIRM) (SEQ ID NO:3) and its encoded amino acid sequence (SEQ ID NO:4).

FIG. 4 Recombinant BpmI endonuclease activity in column fractions following heperin Sepharose chromatography. Lane 1: purified native BpmI endonuclease; lanes 2 to 23: heperin Sepharose column fractions. Fractions 11 to 14 gave rise to complete BpmI digestion of λ DNA. The remaining fractions contain no or partial BpmI activity. Lane 24: 1 kb DNA size marker.

The method described herein by which the BpmI methylase gene and the BpmI restriction endonuclease genes are preferably cloned and expressed in E. coli employ the following steps:

1. Preparation of genomic DNA and restriction digestion of genomic DNA.

Genomic DNA is prepared from Bacillus pumilus (New England Biolabs collection #711) by the standard procedure. Five μg genomic DNA is digested partially with 2, 1, 0.5, and 0.25 units of ApoI (recognition sequence R/AATTY). Genomic DNA fragments in the range of 2-10 kb are purified through a low-melting agarose gel. Genomic and pBR322 DNA are also digested with AatII, BamHI, ClaI, EagI, EcoRI, HindIII, NdeI, NheI, SaI, and SphI, respectively, however, no methylase positive clones were obtained.

2. Construction of ApoI partial genomic DNA library and challenge of library with BpmI.

The ApoI partial DNA fragments are ligated to EcoRI digested and CIP treated pBR322 vector. The ligated DNA is transferred into E. coli RR1 competent cells by electroporation. Transformants are pooled and amplified. Plasmid DNA is prepared from the cells and challenged with BpmI. Following BpmI digestion, the challenged DNA is transformed into RR1 cells. Survivors are screened for resistance to BpmI digestion. Two resistant clones, #18 and #26, were identified to be resistant to BpmI digestion. AatII, BamHI, ClaI, EagI, EcoRI, HindIII, NdeI, NheI, SalI, and SphI digested genomic DNA were also ligated to pBR322 with compatible ends and genomic DNA libraries are constructed. However, no apparent BpmI resistant clones were discovered from these libraries.

3. Subcloning and DNA sequencing of the resistant clone.

One resistant clone #26 contained an insert of about 3.1 kb. The forward and reverse primers of pUC19 were used to sequence the insert. Three ApoI and one HindIII fragments were subcloned in pUC19 and sequenced. The entire insert was sequenced by primer walking. A methylase gene with high homology to amino-methyltransferase is found within the insert which is name BpmI M1 gene. The BpmIM1 gene is 1,650 bp, encoding a 549-amino acid protein with predicted molecular mass of 63,702 daltons.

4. Cloning of BpmI restriction endonuclease gene (BpmIRM) by inverse PCR.

In accordance with the present invention, it was determined that there was one partial open reading frame upstream of BpmIM1 gene that has 31% amino acid sequence identity to another restriction enzyme Eco57I with similar recognition sequence (Eco57I recognition sequence: 5′CTGAAG N16/N14; A. Janulaitis et al. Nucl. Acids Res. 20:6051-6056 (1992); BpmI recognition sequence: 5′CTGGAG N16/N14). Genomic DNA is digested with restriction enzymes. The digested DNA is ligated at a low DNA concentration and then used for inverse PCR amplification of BpmIR gene. Inverse PCR products are derived, gel-purified from low-melting agarose and sequenced. After four rounds of inverse PCR reactions, an open reading frame of 3,030 bp was found upstream of BpmI M1 methylase gene, which encoded a 1,009-amino acid protein with predicted molecular mass of 116,891 daltons. This is one of the largest restriction enzyme discovered so far. By amino acid sequence comparison of BpmI endonuclease with all known proteins in GenBank protein database, it is discovered that BpmI endonuclease is a fusion of two distinct elements with a possible structural domains of restriction-methylation-specificity (R-M-S). This domain organization is analogous to the type I restriction-modification system with three distinct subunits, restriction, methylation, and specificity (R, M, and S). Because BpmI is quite distinct to other type IIs restriction enzymes, it is suggested that BpmI belongs to a subgroup of type II restriction enzymes called type IIf (f stands for fusion of restriction-modification-specificity domains).

5. Expression of BpmIM1 gene in E. coli.

Two primers are synthesized to amplify BpmIM1 gene in PCR. Following digestion with BamHI and SphI, the PCR product is ligated into pACYC184 with the compatible ends. The ligated DNA is transformed into ER2566 competent cells. Plasmids with BpmIM1 gene inserts are tested for resistance to BpmI digestion. Two out of 18 clones were found to be resistant to BpmI digestion, indicating efficient BpmI M1 expression in E. coli cells and BpmI site modification on the expression plasmid. The host cell ER2566 [pACYC-BpmIM1] is used for expression of BpmIRM gene.

BpmI M1 methylase also modifies XhoI site. XhoI recognition sequence 5′CTCGAG3′ is similar to BpmI recognition sequence 5′CTGGAG3′ with only one base difference. It is concluded that BpmI M1 methylase may recognize the sequence 5′CTNNAG3′ and modify the adenine base to generate N6-adenine in the symmetric sequence.

6. Expression of BpmIRM gene in E. coli using a T7 expression vector.

The 3,030-bp BpmIRM gene was amplified in PCR using Taq DNA polymerase, digested with BamHI and ligated into BamHI-digested T7 expression vectors pAII17 and pET21a. After transformation of the ligated DNA into ER2566 [pACYC-BpmIM1], transformants were screened for the endonuclease gene insert. Seven out of 72 clones contained the insert with correct orientation. However, no BpmI activity was detected in cell extracts of IPTG-induced cells. This is probably due to mutations introduced during the PCR process.

To reduce the mutation frequency, Deep Vent® DNA polymerase was used in PCR reactions to amplify the 3030-bp BpmIRM gene. The PCR product was digested with BamHI and XbaI and ligated to T7 expression vectors pAII17 and pET21at. Eighteen out of 36 clones contain the correct size insert. Ten ml cell culture for all 18 clones were induced with IPTG and cell extracts were prepared and assayed for BpmI activity. Clone #4 displayed partial BpmI activity.

7. Partial purification of recombinant BpmI activity.

Five hundred ml of cell culture was made for the expression clone #4 ER2566 [pACYC-BpmIM1, pET21at-BpmIRM]. Cell extract (40 ml) containing BpmI was purified through a heparin Sepharose column. Proteins were eluted with a NaCl gradient of 50 mM to 1 M. Fractions 6 to 27 are assayed for BpmI activity on λ DNA. It was found that fractions 15 to 18 contained the most active BpmI activity (FIG. 4). The yield was estimated at 1,800 units of BpmI per gram of wet E. coli cells. The specific activity was estimated at 24,000 units per mg of protein.

The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.

The references cited above and below are hereby incorporated by reference herein.

1. Preparation of genomic DNA and restriction digestion of genomic DNA.

Genomic DNA is prepared from Bacillus pumilus (New England Biolabs collection #711) by the standard procedure consisting of the following steps:

(a) cell lysis by addition of lysozyme (2 mg/ml final), sucrose (1% final), and 50 mM Tris-HCl, pH 8.0;

(b) cell lysis by addition of 10% SDS (final concentration 0.1%);

(c) cell lysis by addition of 1% Triton X-100 and 62 mM EDTA, 50 mM Tris-HCl, pH 8.0;

(d) phenol-CHCl₃extraction of DNA 3 times (equal volume) and CHCl₃extraction one time;

(e) DNA dialysis in 4 liters of TE buffer, change 3×; and

(f) RNA was removed by RNAse A treatment and the genomic DNA was precipitated in ethanol and resupended in TE buffer;

Five μg genomic DNA was digested partially with 2, 1, 0.5, and 0.25 units of ApoI (recognition sequence R/AATTY) at 50° C. for 30 min. Genomic DNA fragments in the range of 2-10 kb were purified through a 1% low-melting agarose gel. Genomic and pBR322 DNA were also digested with AatII, BamHI, ClaI, EagI, EcoRI, HindIII, NdeI, NheI, SalI, and SphI, respectively. Genomic DNA fragments were ligated to pBR322 with compatible ends.

2. Construction of ApoI partial genomic DNA library and challenge of library with BpmI.

The Apol partial DNA fragments were ligated to EcoRI digested and CIP treated pBR322 vector. The ligated DNA was dialyzed by drop dialysis on 4 L of distilled water and transferred into E. coli RR1 competent cells by electroporation. Ap^Rtransformants were pooled and amplified. Plasmid DNA was prepared from the overnight cells and challenged with BpmI. Following BpmI digestion, the challenged DNA was transformed into RR1 cells. Ap^Rsurvivors were screened for resistance to BpmI digestion. A total of 36 plasmid mini-preparations were made. Two resistant clones, #18 and #26, were identified to be resistant to BpmI digestion. AatII, BamHI, ClaI, EagI, EcoRI, HindIII, NdeI, NheI, SalI, and SphI digested genomic DNA were also ligated to pBR322 with compatible ends and genomic DNA libraries were constructed. However, no apparent BpmI resistant clones were discovered from these libraries after screening more than 144 clones.

3. Subcloning and DNA sequencing of the resistant clone.

One resistant clone #26 contains an insert of about 3.1 kb. The forward and reverse primers of pUC19 were used to sequence the insert. Three ApoI and one HindIII fragments were gel-purified and subcloned in pUC19 and sequenced. The rest of the insert was sequenced by primer walking. A methylase gene with high homology to amino-methyltransferase (N6-adenine methylase) was found within the insert which was name BpmI M1 gene. The BpmIM1 gene is 1,650 bp, encoding a 549-amino acid protein with predicted molecular mass of 63,702 daltons.

4. Cloning of BpmI restriction endonuclease gene (BpmIRM) by inverse PCR.

There is one partial open reading frame upstream of BpmIM1 gene that has 31% amino acid sequence identity to another restriction enzyme Eco57I with similar recognition sequence (Eco57I recognition sequence: 5′CTGAAG N16/N14; A. Janulaitis et al. Nucl. Acids Res. 20:6051-6056 (1992); BpmI recognition sequence: 5′CTGGAG N16/N14). Genomic DNA was digested with restriction enzymes AseI, BclI, HaeII, HpaII, MboI, MseI, NlaIII, PacI, and Tsp509I. The digested DNA was ligated at a low DNA concentration at 2 μg/ml and then used for inverse PCR amplification of BpmIR gene. The sequence of the inverse PCR primers was the following:

5′ gtggaaacggaccgtattatggtt 3′ (232-34) (SEQ ID NO:5)

5′ caccagtaaataacaggttattcc 3′ (232-35) (SEQ ID NO:6)

Inverse PCR conditions were 94° C. 1 min, 55° C. 1 min, 72° C. 2 min for 35 cycles. Inverse PCR products were derived from HaeIII and NlaIII templates, gel-purified from low-melting agarose and sequenced using primers 232-34 and 35.

The primers for second round of inverse PCR were the following:

5′ ttcgtagcaagtacggtccatatcagt 3′ (233-76) (SEQ ID NO:7)

5′ ccgtatgtacttgataggaataacctg 3′ (233-77) (SEQ ID NO:8)

Genomic DNA was digested with AseI, BclI, BsrFI, BstNI, EcoRI, HincII, HindIII, HpaII, NcoI, PacI, PvuI, TaqI, TfiI, and XbaI. The digested DNA was ligated at a low DNA concentration at 2 μg/ml and then used for inverse PCR amplification of BpmIR gene. Inverse PCR conditions were 94° C. 1 min, 55° C. 1 min, 72° C. 2 min for 35 cycles. Inverse PCR products were derived from AseI, HindIII, HpaII, and TaqI templates, gel-purified from low-melting agarose and sequenced using primers 233-76 and 77.

The primers for third round of inverse PCR were the following:

5′ aggaactaagaaagttcatagctg 3′ (234-61) (SEQ ID NO:9)

5′ atgcggtattatataacccaacag 3′ (234-62) (SEQ ID NO:10)

Genomic DNA was digested with AflIII, BspHI, BstNI, EcoRI, HaeII, HinP1I, HhaII, HindIII, StyI, and XmnI. The digested DNA was ligated at a low DNA concentration at 2 μg/ml and then used for inverse PCR amplification of BpmIR gene. Inverse PCR conditions were 94° C. 1 min, 55° C. 1 min, 72° C. 2 min for 35 cycles. Inverse PCR products were derived from HinP1I and XmnI templates, gel-purified from low-melting agarose and sequenced using primers 234-61 and 62.

The primers for the fourth round of inverse PCR were the following:

5′ tgacgtcctcttcacctaattcgg 3′ (235-50) (SEQ ID NO:11)

5′ gagtttgtgaagatagaaccattg 3′ (235-51) (SEQ ID NO:12)

Genomic DNA was digested with ApoI, BstBI, BstYI, ClaI, EcoRI, NdeI, RsaI, Sau3AI, SspI, TaqI, and XmnI. The digested DNA was ligated at a low DNA concentration at 2 μg/ml and then used for inverse PCR amplification of BpmIR gene. Inverse PCR conditions were 94° C. 1 min, 55° C. 1 min, 72° C. 2 min for 35 cycles. Inverse PCR products were derived from ApoI, ClaI, NdeI, RsaI, SspI, and TaqI templates, gel-purified from low-melting agarose and sequenced using primers 235-50 and 51. The ClaI fragment (2.4 kb) further extends upstream of BpmIRM gene. The rest of the ClaI fragment was sequenced using primer walking.

After four rounds of inverse PCR reactions, an open reading frame of 3,030 bp was found upstream of BpmI M1 methylase gene, which encodes a 1,009-amino acid protein with predicted molecular mass of 116,891 daltons. This is one of the largest restriction enzyme discovered so far. By amino acid sequence comparison of BpmI endonuclease with all known proteins in GenBank protein database, it was discovered that BpmI endonuclease is a fusion of two distinct elements with a possible structural domains of restriction-methylation-specificity (R-M-S). This domain organization is analogous to the type I restriction-modification system with three distinct subunits, restriction, methylation, and specificity (R, M, and S). Because BpmI is quite distinct to other type IIs restriction enzymes, it is proposed that BpmI belongs to a subgroup of type II restriction enzymes called type IIf (f stands for fusion of restriction-modification-specificity domains)

5. Expression of BpmIM1 gene in E. coli.

Two primers are synthesized to amplify BpmIM1 gene in PCR. The primer sequences are:

forward:

5′ agcggatccggaggtaaataaatgaatcaattaattgaaaatgttaat 3′ (238-177) (SEQ ID NO:13)

reverse:

5′ aagggggcatgcttatacttatttcttcgttctattgtttct 3′ (238-178) (SEQ ID NO:14)

Following digestion with BamHI and SphI, the PCR product was ligated into pACYC184 with the compatible ends. The ligated DNA was transformed into ER2566 competent cells. Cm^Rtransformants were plated at 37° C. overnight. Plasmids with BpmIM1 gene inserts were tested for resistance to BpmI digestion. Two out of 18 clones showed full resistance to BpmI digestion, indicating efficient BpmI M1 expression in E. coli cells and BpmI site modification on the expression plasmid. The host cell ER2566 [pACYC-BpmIM1] was used for expression of BpmIRM gene.

BpmI M1 methylase also modifies XhoI site. XhoI recognition sequence 5′CTCGAG3′ is similar to BpmI recognition sequence 5′CTGGAG3′ with only one base difference. It is concluded that BpmI M1 methylase may recognize the sequence 5′CTNNAG3′ and modify the adenine base to generate N6-adenine in the symmetric recognition sequence.

6. Expression of BpmIRM gene in E. coli using a T7 expression vector.

Two primers were synthesized to amplify the BpmIRM gene. The primer sequences were:

5′ caaggatccggaggtaaataaatgcatataagtgagttagtagataaatac 3′ (247-217) (SEQ ID NO:15)

5′ ttaggatcctcatttttcttctcctaacgccgctgt 3′ (238-182) (SEQ ID NO:16)

The 3,030-bp BpmIRM gene was amplified in PCR using Taq DNA polymerase, digested with BamHI and ligated into BamHI-digested T7 expression vectors pAII17 and pET21a. After transformation of the ligated DNA into ER2566 [pACYC-BpmIM1], Ap^RCm^Rtransformants were screened for the endonuclease gene insert. Seven out of 72 clones contained the insert with correct orientation. However, no BpmI activity was detected in cell extracts of IPTG-induced cells. This was probably due to mutations introduced during the PCR process.

To reduce the mutation frequency, Deep Vent® DNA polymerase was used in PCR reactions to amplify the 3,030-bp BpmIRM gene. The forward primer incorporated an XbaI site and its sequence is the following:

5′ caccaatctagaggaggtaaataaatgcatataagtgagttagtagataaatac 3′ (238-181) (SEQ ID NO:17)

PCR was performed using primers 238-181, 238-182, and Deep Vent® DNA polymerase. The PCR conditions were 94° C. 5 min for one cycle; 94° C. 1 min, 55° C. 1.5 min, 72° C. 8 min for 20 cycles. The PCR product was purified through a Qiagen spin column and digested with BamHI and XbaI and ligated to T7 expression vectors pAII17 and pET21 at with compatible ends. Eighteen out of 36 clones contain the correct size insert. Ten ml cell culture for all 18 clones containing inserts were induced with IPTG for 3 h and cell extracts were prepared by sonication and assayed for BpmI activity. Clone #4 displayed partial BpmI activity. Because this gene was derived by PCR cloning, the entire BpmIRM fusion gene was sequenced on both strands and it was confirmed to be wild type sequence.

7. Partial purification of recombinant BpmI activity.

Five hundred ml of cell culture was made for the expression clone #4 ER2566 [pACYC-BpmIM1, pET21at-BpmIRM]. The late log cells were induced with IPTG and Cell extract (40 ml) containing BpmI was purified through a heparin Sepharose column. Proteins were eluted with a NaCl gradient of 50 mM to 1 M. Fractions 6 to 27 contained the most protein concentration and were assayed for BpmI activity on λ DNA. It was found that fractions 15 to 18 contained the most active BpmI activity (FIG. 4). The yield was estimated at 1,800 units of BpmI per gram of wet E. coli cells. The specific activity was estimated at 24,000 units per mg of protein. Proteins from fractions 15 to 18 were analyzed on a SDS-PAGE gel and protein bands were stained with Gelcode blue stain. A protein band corresponding to ˜115 kDa was detected on the protein gel, in close agreement with the predicted size of 117 kDa.

The E. coli strain ER2566 [pACYC-BpmIM1, pET21at-BpmIRM] has been deposited under the terms and conditions of the Budapest Treaty with the American Type Culture Collection on Oct. 12, 2000 and received Accession No. PTA-2598.

Two primers were synthesized to amplify the putative endonuclease domain with deletion of the methylase and specificity domains. The deletion clone thus contains only the R portion and the M and S portions were removed. The forward primer was 238-181 as described above. The reverse primer had the following sequence with a XhoI site at the 5′ end:

5′ tgaaatctcgagttatcctgatccacaacatatatctgctat 3′ (244-95) (SEQ ID NO:18)

The deletion junction was in motif I of γ type N6 adenine methylase. The γ type N6 adenine methylases contain conserved motifs of X, I, II, III, IV, V, VI, VII, VIII. The specificity domain (TRD) is located after motif VIII. The BpmI deletion clone (BpmI-Δ#1) still carried motifs X and part of motif I. The specificity domain after motif VIII was also deleted (the remaining portion is shown in FIG. 1).

PCR was performed using primers 238-181 and 244-95 and Taq plus Vent® DNA polymerase (94° C. 1 min, 60° C. 1 min, and 72° C. 1 min for 25 cycles). The PCR product was digested with XbaI and XhoI and cloned into a T7 expression vector pET21b. Sixteen clones out of 36 screened contained the correct size insert and the cells were induced with IPTG for 3 h. Cell extract was prepared by sonication and assayed for BpmI activity on λ DNA. However, no apparent BpmI digestion pattern was detected. Only non-specific nuclease was detected in cell extract, resulting in a smearing of DNA substrate. It was concluded that deletion of the methylase and specificity portion of the BpmIRM fusion protein abolished BpmI restriction activity.

To further confirm the above result, another deletion clone was constructed that deleted methylase motifs IV, V, VI, VII, VIII, and the specificity domain. This EcoRI fragment deletion mutant contains 1,521 bp (507 amino acid) deletion at the C-terminus half of the fusion protein (BpmI-Δ#2). IPTG-induced cell extract of this mutant also did not display BpmI endonuclease activity.

To delete the specificity domain (target-recognizing domain, TRD), a HindIII fragment of 579 bp (193 amino acid) was deleted from the C-terminus of BpmI RM fusion endonuclease (BpmI-Δ#3). IPTG-induced cell extract of the TRD deletion mutant did not show any BpmI endonuclease activity. However, the mutant protein displayed non-specific nuclease activity. It was concluded that the specificity (TRD) domain is also required for BpmI endonuclease activity. Deletion of the specificity (TRD) domain may abolish or reduce its DNA binding affinity and specificity. By swapping in of other N6 methylase and specificity domains, one may be able to create new enzyme specificity.

Since BpmI endonuclease consists of three domains (R-M-S), it is possible to plug in other methylation-specificity domains to create a new enzyme specificity. The BpmIRM fusion gene is cloned in a T7 expression vector as described in Example 1. Plasmid DNA is prepared. The γ type N6 adenine methylases contain conserved motifs of X, I, II, III, IV, V, VI, VII, VIII (Malone T. et al. J. Mol.Biol 253:618-632 (1995)). Motifs X through VIII and TRD are deleted and a DNA linker coding for one or more bridging amino acids is inserted with a restriction site, preferably blunt (for example SmaI site). The number of amino acids will differ from one system to the next and can be determined by routine experimentation. The goal is to provide sufficient steric space for the introduction of the new M-S domains. DNA coding for other γ type N6 adenine methylases containing motifs of X, I, II, III, IV, V, VI, VII, VIII and TRD are ligated to the digested blunt site (in frame) of the BpmI deletion clone. The ligated DNA is transformed into a non-T7 expression vector. After the insert is verified, the plasmid containing new methylation-specificity domains is transformed into a T7 expression host and induced with IPTG. Cell extract is assayed on plasmid and phage DNA and analyzed for new restriction activity.