New England Biolabs, Inc.

Jian-ping Xiao

Shuang-Yong Xu

Zhenyu Zhu

Robert E Maunus

The present invention relates to recombinant DNA which encodes the BpmI restriction endonuclease as well as BpmI methyltransferase, expression of BpmI restriction endonuclease from 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)).

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

The present invention relates to recombinant DNA which encodes the NheI restriction endonuclease as well as NheI methyltransferase, expression of NheI restriction endonuclease from cells containing the recombinant DNA. An internal NdeI site in the nheIR gene was eliminated by a silent mutation. A new NdeI site was engineered at the start codon of nheIR gene. An NdeI-BamHI fragment containing nheIR gene was cloned into a T7 expression vector pAII17 and expressed in a premodified host ER2566 &lsqb;pACYC-NheIM, pAII17-NheIR2&rsqb;. The recombinant clone produces approximately 10 million units of NheI per gram of wet cells.

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

The present invention relates to recombinant DNA molecules encoding NspHI restriction endonuclease and methylase and to method to use premodified E. coli K strain RR1 (.lambda.DE3) for overexpression of NspHI restriction endonuclease.

Method for cloning the NSPHI restriction-modification system in E. coli

BsrFI restriction enzyme was purified from Bacillus stearothermophilus to near homogeneity. The protein was sequenced to obtain its N-terminus amino acid sequence. A set of denegerate primers were synthesized based on the aa sequence. The first 18 codons encoding BsrFI restriction endonuclease (bsrFIR) was amplified by PCR and its coding sequence was obtained. The methylase selection method was used to clone BsrFI methylase gene (bsrFIM). Two clones were found to be resistant to BsrFI digstion. The entire insert in one clone was sequenced and the insert encodes the BsrFI methylase (M. BsrFI). In addition, a small truncated open reading frame adjacent to the methylase gene has homology to Cfr10I restriciton endonuclease in a BlastX homology search in Genbank database. BsrFI and Cfr10I are isoschizomer that recognizes and cleaves 5'R CCGGY3'. Two primers were used to amplify the bsrFIR gene, The forward primer is a degenerate primer designed from the N-terminus aa sequence and the reverse primer is the bona fide sequence derived from the BsrFI methylase.sup.+ clone. The bsrFIR gene was amplified by PCR, ligated into a T7 expression vector pET21at and the ligated DNA was transformed into premodified cells ER2566 [pLG339-BsrFIM]. The final expression strain is ER2566 [pLG339-BsrFIM, pET21at-BsrFIR]. Recombinant BsrFI activity was detected in E. coli cell extract. BsrFI is cloned from a thermophile Bacillus stearothermophilus. Thus, BsrFI a thermostable enzyme and it is active at 37.degree. C. to 65.degree. C.

Method for cloning and expression of BsrFI restriction endonuclease in

The methylase selection method was used to clone the BslI methylase gene (bslIM) from Bacillus species. A partially active BslI methylase lacking the 17 amino acid residues at the N-terminus was cloned in E. coli using expression vector pRRS. Inverse PCR was used to clone the missing portion of the BslI methylase. After cloning the complete BslI methylase gene and its upstream DNA sequences, a RadC homolog was found upstream of the BslI methylase. Because methylase gene and restriction endonuclease gene are located in proximity to each other in a particular restriction-modification system, efforts were made to clone the downstream DNA by inverse PCR. After two round of inverse PCR reactions two open reading frames (ORF) were found downstream of the BslI methylase gene. Expression of the first ORF (ORF1) in a T7 expression vector did not yield any active BslI endonuclease. Expression of the second ORF (ORF2) in E. coli and assay of the crude cell extract indicated that this gene product has DNA nicking activity. The gene product of ORF2 alone does not constitute BslI endonuclease activity. Expression of ORF1 and ORF2 in the same E. coli cell produces BslI endonuclease activity. BslI endonuclease activity can be reconstituted in vitro by mixing gene product of ORF1 and ORF2 together.

Method for cloning and producing the BS1I restriction endonuclease in E.

The present invention relates to cloning recombinant DNA molecules encoding a multi-specific methylase gene (bssHIIM1), BssHII restriction endonuclease gene (bssHIIR), and the cognate BssHII methylase gene (bssHIIM2) from Bacillus stearothermophilus H3 E. coli. The BssHII multi-specific methylase gene was first cloned in a Sau3AI library using a modified pLITMUS28 vector (New England Biolabs, Inc., Beverly, Mass.) with two BssHII sites. Expression of the multi-specific BssHII methylase renders the two BssHII sites resistant to BssHII digestion. Surprisingly, the cloned methylase also modifies some other sites in addition to BssHII site (5'GCGCGC3'). The methylase also modifies BsrFI site (5'RCCGGY3') and HaeII site (5'RGCGCY3'); and partially modifies EagI site (5'CGGCCG3') and MIul site (5'ACGCGT3'). The beginning of the bssHIIR gene was cloned by using two degenerate primers based on the N-terminal amino acid sequence in PCR. The rest of the bssHIIR gene was cloned by inverse PCR. The cognate bssHIIM2 gene was cloned by inverse PCR and PCR. The BssHII restriction endonuclease gene was expressed in E. coli host ER417 carrying three plasmids pLysP, pLG-BssHIIM2, pET21AT-BssHIIR.

Method for cloning and producing the bssHII restriction endonuclease in

The present invention relates to isolated DNA coding for the restriction endonuclease SCaI as well as to a method for cloning methylase genes from Streptomyces into E. coli by a modification of the methylase selection method. At first, the standard methylase gene selection method was tried to clone the SCaI methylase gene using a high-copy-number cloning vector pUC19 during library construction. The SCaI methylase gene was refractory to cloning by using pUC19, presumably due to the poor expression of the SCaI methylase gene in E. coli. If the SCaI methylase is not efficiently expressed in E. coli, the SCaI sites on the plasmid will not be sufficiently modified by the methylase. As a consequence, the plasmid will be cleaved and lost in the plasmid library after SCaI endonuclease challenge. Since the standard methylase selection did not work, the 'endo-blue method' was tried to clone the SCaI endonuclease gene. Nineteen blue colonies were identified, but none of them yielded any detectable SCaI endonuclease activity. The SCaI endonuclease gene was first cloned by inverse PCR using primers that annealed to the end of the SCaI methylase gene. In order to increase the SCaI endonuclease expression in E. coli, an optimal ribosome binding site and spacing were engineered in front of the ATG start codon and the gene was inserted into expression vector pRRS.

Method for cloning and producing the SCaI restriction endonuclease in E.

The present invention relates to recombinant DNA which encodes the SapI restriction endonuclease and modification methylase, and the production of SapI restriction endonuclease from the recombinant DNA as well as to methods for cloning Actinomycetes genes into suitable hosts such as E. coli.

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Wenham, MA, United States of America

Jian-ping Xiao