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

摘要

Rsal, a restriction enzyme from the bacterium <Italic>Rhodopseudomonas sphaeroides,</Italic> recognizes the DNA sequence 5'-GTAC-3'.Because RsaI is commercially valuable, we sought to overproduce it by cloning the genes for Rsal and its accompanying, modification, enzyme. The 'methylase-selection' method, the customary procedure for cloning restriction and modification genes, was applied to RsaI. The method yielded clones containing the methylase gene (<Italic>rsa</Italic>IM), but none containing both the methylase gene and the restriction gene (<Italic>rsa</Italic>IR). Inverse-PCR was then used to recover sections of the DNA downstream of <Italic>rsa</Italic>IM. These sections were sequenced, and the sequences were joined <Italic>in silico</Italic> to reveal the gene organization of the RsaI R-M system. By comparing the coding potential of the DNA with the N-terminal amino acid sequence of the purified Rsal restriction enzyme, we discovered that the Rsal R and M genes, rather than being adjacent-the situation that pertains in most R-M systems-are separated by an intervening gene of unknown function.

Based on this information, the rsaIR gene was cloned by PCR instead of methylase-selection. These new clones proved to be highly unstable, however, even in the presence of the rsalM gene. Various attempts were made to stabilize the gene, but most met with failure. Stability was finally achieved by introducing a second methylase gene, mjaVM, to augment the protection provided by rsaIM, and by tightly controlling the expression of rsaIR using a special two-promoter, anti-sense transcription, expression vector.