Method for cloning and producing the SnaBI restriction endonucle

Details Method for cloning and producing the SnaBI restriction endonucle Method for cloning and producing the SnaBI restriction endonucle

1998-08-31

2000-02-15

Patterson, Jr., Charles L.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

4352523, 4353201, 536 232, C12N 922, C12N 1555

Patent

active

060251794

ABSTRACT:
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 a restriction-modification systems, efforts were made to clone the downstream DNA by inverse PCR. Inverse PCR cloned the missing portion of the SnaBI endonuclease and also identified a control, or C, protein. The two open reading frames snaBIR (ORF1) and snaBIC (ORF2) converged toward 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.

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