Method for cloning and expression of BpmI restiction endonuclease in E. coli

Abstract: The present invention relates to recombinant DNA which encodes the Bpm I restriction endonuclease as well as Bpm I methyltransferase, expression of BpmI restriction endonuclease from E. coli cells containing the recombinant DNA. Bpm I endonuclease is a fusion of two distinct elements with a possible structural domains of restriction-methylationspecificity (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 Eco 57I, Bpm I, Gsu I, Bse RI and some other restriction enzymes that cut downstream sequences at long distance, 10-20 bp downstream of recognition sequence, such as Mme I (N20/N18)).

Description: BACKGROUND OF THE INVENTION The present invention relates to recombinant DNA which encodes the Bpm I restriction endonuclease as well as Bpm I methyltransferase and expression of Bpm I restriction endonuclease from E. coli cells containing the recombinant DNA. Bpm I is an isoschizomer of Gsu I (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 Dra I, Dra II and Dra III. These enzymes recognize and cleave the sequences 5TTT/AAA3', 5'PuG/GNCCPy3' and 5'CACNNN/GTG3' respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, Eco RI, 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 -3 to 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 ( Eco RII: Kosykh et al., Mol. Gen. Genet. 178:717-719 (1980); Hha II: Mann et al., Gene 3:97-112 (1978); Pst I: 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 ( Eco RV: 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); Pvu II: Blumenthal et al., J . Bacteriol . 164:501-509 (1985); Tsp 45I: Wayne et al. Gene 202:83-88 (1997)). A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Patent No. 5,200,333 and Bsu RI: 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 ( Bsp RI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); Bsu RI: Kiss and Baldauf, Gene 21:111-119 (1983); and Msp I: 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. Patent 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 ( Taq I, Tth 111I, Bso BI, Tf nuclease) have been cloned by this method (U.S. Patent 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. SUMMARY OF THE INVENTION The present invention relates to a method for cloning the Bpm I restriction endonuclease from Bacillus pumilus into E.coli by methylase selection and inverse PCR amplification of the adjacent DNA of the Bpm I methylase gene. The present invention relates to recombinant Bpm I and methods for producing the same. Bpm I restriction endonuclease is found in the strain of Bacillus pumilus (New England Biolabs' strain collection #711). It recognizes doublestranded 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 Eco 57I with similar recognition sequence ( Eco 57I 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 Bpm I M1 methylase gene, which encodes a 1009-aa protein with predicted molecular mass of 116,891 daltons. By amino acid sequence comparison of Bpm I endonuclease with all known proteins in GenBank protein database, it was discovered that Bpm I 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 Bpm I is quite distinct to other type IIs restriction enzymes, it is proposed that Bpm I 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. Bpm I M1 methylase also modifies Xho I site. Xho I recognition sequence 5' CT C GAG 3' is similar to Bpm I recognition sequence 5' CT G GAG 3' with only one base difference. It was concluded that Bpm I M1 methylase may recognize the sequence 5' CTNN A G 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 product. The BpmIRM gene was first amplified by Taq DNA polymerase and cloned into the premodified host, but no Bpm I 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 Bpm I activity. This clone was sequenced and confirmed to contain wild type sequence. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Gene organization of Bpm I restriction-modification system. Genes BpmIRM and BpmIM1 code for BpmI endonuclease ( Bpm I endonuclease-methylase fusion protein and Bpm I M1, respectively. Bpm I-Δ#1, Bpm I-Δ#2, and Bpm I-Δ#3 are deletion mutants with deletions in the methylation or specificity domains. Figure 2 DNA sequence of Bpm I M1 methylase gene ( BpmIM1 ) (SEQ ID NO:1) and its encoded amino acid sequence (SEQ ID NO:2). Figure 3 DNA sequence of Bpm I endonuclease gene ( BpmIRM ) (SEQ ID NO:3) and its encoded amino acid sequence (SEQ ID NO:4). Figure 4 Recombinant Bpm I endonuclease activity in column fractions following heperin Sepharose chromatography. Lane 1: purified native Bpm I endonuclease; lanes 2 to 23: heperin Sepharose column fractions. Fractions 11 to 14 gave rise to complete Bpm I digestion of λ DNA. The remaining fractions contain no or partial Bpm I activity. Lane 24: 1 kb DNA size marker. DETAILED DESCRIPTION OF THE INVENTION The method described herein by which the Bpm I methylase gene and the Bpm I 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 Apo I (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 Aat II, Bam HI, Cla I, Eag I, Eco RI, Hin d III, Nde I, Nhe I, Sal I, and Sph I, respectively, however, no methylase positive clones were obtained. 2. Construction of Apo I partial genomic DNA library and challenge of library with Bpm I. The Apo I partial DNA fragments are ligated to Eco RI 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 Bpm I. Following Bpm I digestion, the challenged DNA is transformed into RR1 cells. Survivors are screened for resistance to Bpm I digestion. Two resistant clones, #18 and #26, were identified to be resistant to Bpm I digestion. Aat II, Bam HI, Cla I, Eag I, Eco RI, Hin dIII, Nde I, Nhe I, Sal I, and Sph I digested genomic DNA were also ligated to pBR322 with compatible ends and genomic DNA libraries are constructed. However, no apparent Bpm I 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 Apo I and one Hin dIII 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 Bpm I 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 Bpm I 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 Eco 57I with similar recognition sequence ( Eco 57I recognition sequence: 5'CTG A AG N16/N14; A. Janulaitis et al. Nucl. Acids Res. 20:6051-6056 (1992); Bpm I 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 Bpm I 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 Bpm I endonuclease with all known proteins in GenBank protein database, it is discovered that Bpm I 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 Bpm I is quite distinct to other type IIs restriction enzymes, it is suggested that Bpm I 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 Bam HI and Sph I, 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 Bpm I digestion. Two out of 18 clones were found to be resistant to Bpm I digestion, indicating efficient Bpm I M1 expression in E. coli cells and Bpm I site modification on the expression plasmid. The host cell ER2566 [pACYC-BpmIM1] is used for expression of BpmIRM gene. Bpm I M1 methylase also modifies Xho I site. Xho I recognition sequence 5'CT C GAG3' is similar to BpmI recognition sequence 5'CT G GAG3' with only one base difference. It is concluded that Bpm I M1 methylase may recognize the sequence 5'CTNN A G3' 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 Bam HI and ligated into Bam HI-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 Bpm I 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 Bam HI and Xba I 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 Bpm I activity. Clone #4 displayed partial Bpm I activity. 7. Partial purification of recombinant Bpm I activity. Five hundred ml of cell culture was made for the expression clone #4 ER2566 [pACYC-BpmIM1, pET21at-BpmIRM]. Cell extract (40 ml) containing Bpm I 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 Bpm I activity on λ DNA. It was found that fractions 15 to 18 contained the most active Bpm I activity (Figure 4). The yield was estimated at 1,800 units of Bpm I 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. EXAMPLE 1 CLONING OF BpmI RESTRICTION-MODIFICATION SYSTEM IN E. COLI 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 the following steps: (a) cell lysis by addition of lysozyme (2 mg/ml final), sucrose (1% final), and 50 mM Tris-HCI, 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-HCI, pH 8.0; (d) phenol-CHCl 3 extraction of DNA 3 times (equal volume) and CHCl 3 extraction one time; (e) DNA dialysis in 4 liters of TE buffer, change 3x; 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 Apo I (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 Aat II, Bam HI, Cla I, Eag I, Eco RI, Hind III, Nde I, Nhe I, Sal I, and Sph I, respectively. Genomic DNA fragments were ligated to pBR322 with compatible ends. 2. Construction of Apo I partial genomic DNA library and challenge of library with Bpm I. The Apo I partial DNA fragments were ligated to Eco RI 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 R transformants were pooled and amplified. Plasmid DNA was prepared from the overnight cells and challenged with Bpm I. Following Bpm I digestion, the challenged DNA was transformed into RR1 cells. Ap R survivors were screened for resistance to Bpm I digestion. A total of 36 plasmid mini-preparations were made. Two resistant clones, #18 and #26, were identified to be resistant to Bpm I digestion. Aat II, Bam HI, Cla I, Eag I, Eco RI, Hind III, Nde I, Nhe I, Sal I, and Sph I digested genomic DNA were also ligated to pBR322 with compatible ends and genomic DNA libraries were constructed. However, no apparent Bpm I 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 Apo I and one Hin dIII 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 Bpm I 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 Bpm I 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 Eco 57I with similar recognition sequence ( Eco 57I recognition sequence: 5'CTG A AG N16/N14; A. Janulaitis et al. Nucl. Acids Res. 20:6051-6056 (1992); Bpm I recognition sequence: 5'CTG G AG N16/N14). Genomic DNA was digested with restriction enzymes Ase I, Bcl I, Hae II, Hpa II, Mbo I, Mse I, Nla III, Pac I , and Tsp 509I. 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: 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 Hae III and Nla III 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: Genomic DNA was digested with Ase I, Bcl I, Bsr FI, Bst NI, Eco RI, Hinc II, Hind III, Hpa II, Nco I, Pac I, Pvu I, Taq I, Tfi I, and Xba I. 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 Ase I, Hind III, Hpa II, and Taq I 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: Genomic DNA was digested with Afl IIII , Bsp HI, Bst NI, Eco RI, Hae II, Hin P1I , Hha II, Hind III, Sty I, and Xmn I. The digested DNA was ligated at a low DNA concentration at 2 µg/ml and then used for inverse PCR amplification of Bpm IR 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 Hin P1I and Xmn I 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: Genomic DNA was digested with Apo I, Bst BI, Bst YI, Cla I, Eco RI, Nde I, Rsa I, Sau 3AI, SspI, Taq I, and Xmn I. 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 Apo I, Cla I, Nde I, Rsa I, Ssp I, and Taq I templates, gel-purified from low-melting agarose and sequenced using primers 235-50 and 51. The Cla I fragment (2.4 kb) further extends upstream of BpmIRM gene. The rest of the Cla I 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 Bpm I 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 Bpm I endonuclease with all known proteins in GenBank protein database, it was discovered that Bpm I 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 Bpm I is quite distinct to other type IIs restriction enzymes, it is proposed that Bpm I 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: reverse: Following digestion with Bam HI and Sph I, the PCR product was ligated into pACYC184 with the compatible ends. The ligated DNA was transformed into ER2566 competent cells. Cm R transformants were plated at 37°C overnight. Plasmids with BpmIM1 gene inserts were tested for resistance to Bpm I digestion. Two out of 18 clones showed full resistance to Bpm I digestion, indicating efficient Bpm I 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. Bpm I M1 methylase also modifies Xho I site. Xho I recognition sequence 5'CT C GAG3' is similar to BpmI recognition sequence 5'CT G GAG3' with only one base difference. It is concluded that BpmI M1 methylase may recognize the sequence 5'CTNN A G3' 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: The 3,030-bp BpmIRM gene was amplified in PCR using Taq DNA polymerase, digested with Bam HI and ligated into Bam HI-digested T7 expression vectors pAII17 and pET21a. After transformation of the ligated DNA into ER2566 [pACYC-BpmIM1], Ap R Cm R transformants were screened for the endonuclease gene insert. Seven out of 72 clones contained the insert with correct orientation. However, no Bpm I 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 Xba I site and its sequence is the following: 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 Bam HI and Xba I and ligated to T7 expression vectors pAII17 and pET21at 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 3h and cell extracts were prepared by sonication and assayed for Bpm I activity. Clone #4 displayed partial Bpm I 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 Bpm I 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 Bpm I 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 Bpm I activity on λ DNA. It was found that fractions 15 to 18 contained the most active Bpm I activity (Figure 4). The yield was estimated at 1,800 units of Bpm I 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 October 12, 2000 and received Accession No. PTA-2598. Example 2 Deletion of the methylase portion of Bpm I RM fusion protein 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 Xho I site at the 5' end: 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 Bpm I deletion clone ( Bpm I-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 Figure 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 Xba I and Xho I 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 3h. Cell extract was prepared by sonication and assayed for Bpm I activity on λ DNA. However, no apparent Bpm I digestion pattern was detected. Only non-specific nuclease was detected in cell extract, resulting a smearing of DNA substrate. It was concluded that deletion of the methylase and specificity portion of the Bpm IRM fusion protin abolished Bpm I 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 Eco RI fragment deletion mutant contains 1,521 bp (507 amino acid) deletion at the C-terminus half of the fusion protein ( Bpm I-Δ#2). IPTG-induced cell extract of this mutant also did not display Bpm I endonuclease activity. To delete the specificity domain (target-recognizing domain, TRD), a Hin dIII fragment of 579 bp (193 amino acid) was deleted from the C-terminus of Bpm I RM fusion endonuclease ( Bpm I-Δ#3). IPTG-induced cell extract of the TRD deletion mutant did not show any Bpm I endonuclease activity. However, the mutant protein displayed non-specific nuclease activity. It was concluded that the specificity (TRD) domain is also required for Bpm I 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. Example 3 Generation of new enzyme specificity using Bpm I RM fusion protein Since Bpm I 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 Sma I 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 Bpm I 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. SEQUENCE LISTING <110> New England Biolabs, Inc. <120> Method for Cloning and Expression of BpmI Restriction Endonuclease in E .coli <130> 43635/EP <160> 18 <170> PatentIn version 3.1 <210> 1 <211> 1650 <212> DNA <213> Bacillus pumilus <220> <221> CDS <222> (1)..(1650) <223> <400> 1 <210> 2 <211> 549 <212> PRT <213> Bacillus pumilus <400> 2 <210> 3 <211> 3030 <212> DNA <213> Bacillus pumilus <220> <221> CDS <222> (1)..(3030) <223> <400> 3 <210> 4 <211> 1009 <212> PRT <213> Bacillus pumilus <400> 4 <210> 5 <211> 24 <212> DNA <213> Bacillus pumilus <400> 5 <210> 6 <211> 24 <212> DNA <213> Bacillus pumilus <400> 6 <210> 7 <211> 27 <212> DNA <213> Bacillus pumilus <400> 7 <210> 8 <211> 27 <212> DNA <213> Bacillus pumilus <400> 8 <210> 9 <211> 24 <212> DNA <213> Bacillus pumilus <400> 9 <210> 10 <211> 24 <212> DNA <213> Bacillus pumilus <400> 10 <210> 11 <211> 24 <212> DNA <213> Bacillus pumilus <400> 11 <210> 12 <211> 24 <212> DNA <213> Bacillus pumilus <400> 12 <210> 13 <211> 48 <212> DNA <213> Bacillus pumilus <400> 13 <210> 14 <211> 42 <212> DNA <213> Bacillus pumilus <400> 14 <210> 15 <211> 51 <212> DNA <213> Bacillus pumilus <400> 15 <210> 16 <211> 36 <212> DNA <213> Bacillus pumilus <400> 16 <210> 17 <211> 54 <212> DNA <213> Bacillus pumilus <400> 17 <210> 18 <211> 42 <212> DNA <213> Bacillus pumilus <400> 18

Claims: An isolated DNA segment coding for the Bpm I restriction endonuclease, wherein the isolated DNA is obtainable from ATCC No. PTA-2598. A recombinant DNA vector into which a DNA segment according to claim 1 has been inserted. An isolated DNA segment coding for the Bpm I restriction endonuclease and Bpm I methylase M1 and M2, wherein the isolated DNA is obtainable from ATCC No. PTA-2598. A cloning vector which comprises the isolated DNA of claim 3. A host cell transformed by the vector of claims 2 or 4. A method of producing recombinant Bpm I restriction endonuclease comprising culturing a host cell transformed with the vector of claims 2 or 4 under conditions for expression of said endonuclease. A method for modifying the specificity of a Bpm I restriction-modification system, said system including DNA coding for a restriction endonuclease and methylase; the method comprising the steps: (a) isolating the DNA segment of claim 3, and deleting the methylation-specificity domain of said Bpm I DNA segment to form a Bpm 1 deletion clone; (b) inserting a DNA linker, coding for an appropriate restriction site and coding for one or more amino acids, at the deletion site of step (a) to form a digested blunt site; and (c) ligating in frame the DNA coding fot other γ type N6 adenine methylases containing motifs of X, I, II, III, IV, V, VI, VII, VIII and TRD to the digested blunt site of the Bmp I deletion clone to form a modified DNA encoding a restriction-modification system.

Inventors: XIAO JIAN-PING, XU SHUANG-YONG, ZHU ZHENYU, XIAO, JIAN-PING, XU, SHUANG-YONG, ZHU, ZHENYU
Applicants: NEW ENGLAND BIOLABS INC, NEW ENGLAND BIOLABS, INC.
Application year: 2001
Main classification: C12N15
IPC codes: C12N9, C12N1, C12N5, C12N15