Method for cloning and producing the BssHII restriction endonuclease in E.coli

摘要

The present invention relates to cloning recombinant DNA molecules encoding a multi-specific methylase gene (bssHIIM1), BssHII restriction endonuclease gene (bssHIIR), and the cognate BssHll methylase gene (bssHIIM2) from Bacillus stearothermophilus H3 E. coli. The BssHII multi-specific methylase gene was first cloned in a Sau3Al library using a modified pLITMUS28 vector (New England Biolabs, Inc., Beverly, MA) with two BssHII sites. Expression of the multi-specific BssHll methylase renders the two BssHll sites resistant to BssHll digestion. Surprisingly, the cloned methylase also modifies some other sites in addition to BssHll site (5'GCGCGC3'). The methylase also modifies BsrFI site (5'RCCGGY3') and HaeII site (5'RGCGCY3'); and partially modifies Eagl site (5'CGGCCG3') and MIuI 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 BssHll restriction endonuclease gene was expressed in E. coli host ER417 carrying three plasmids pLysP, pLG-BssHIIM2, pET21AT-BssHIIR.