Method for cloning and producing the snaBI restriction endonuclease and purification thereof

摘要

The methylase selection method was used to clone the SnaBI methylase gene (snaBIM) from Sphaerotilus natans (ATCC 15291). An active SnaBI methylase was cloned in E. coli using pSnaBI-2, a pUC19 derivative containing two SnaBI sites. Because methylase and restriction genes are usually located alongside each other in restriction-modification systems, efforts were made to clone the downstream DNA by inverse PCR. The missing portion of the SnaBI endonuclease was cloned by inverse PCR. A control, or C, protein was identified using the same technique. The two open reading frames snaBIR (ORF1) and snaBIC (ORF2) were orientated towards the SnaBI methylase gene (ORF). Attempts to establish a snaBIR-recombinant plasmid expressing the SnaBI endonuclease in E.coli modified with SnaBI methylase failed. Overexpression of the SnaBI endonuclease in E. coli required the use of the heterospecific BsaAI methylase. <IMAGE>