Screening method of methylation-protected strain for expressing restriction enzyme KasI

Abstract

The invention discloses a screening method of a methylation-protected strain for expressing a restriction enzyme KasI. The method comprises the steps that a gene of methyltransferase M. HhaI is obtained by using a specific genetic primer of the methyltransferase M. HhaI from the genome of a strain Haemophilus haemolyticus; and then a recombinant expression strain of the methyltransferase M. HhaI is constructed, recombinant expression strains of the restriction enzyme KasI are screened and constructed, finally recombinant expression plasmid pBAD-R. KasI transforms competent cells for screeningand culture, and the recombinant expression strain of the restriction enzyme KasI is obtained. According to the method, highly efficient recombinant expression of the restriction enzyme KasI is realized; the recombinant expression of the restriction enzyme KasI is performed by using an arabinose operon expression system; and the resistance to digestion of host genome DNA by the restriction enzymeKasI is achieved, and the screened M. HhaI methylation-protected strain can further be applied to the recombinant expression of other restriction enzymes with similar recognition sites.

Description

technical field [0001] The invention relates to a screening method for strains, in particular to a screening method for methylation-protected bacterial strains expressing restriction endonuclease KasI. Background technique [0002] Restriction endonucleases are nucleases that can recognize specific sequences in double-stranded DNA sequences and cut them (Ch.8, eds. De Bruijin, et al., Chapman & Hall, New York, 78-92.1998), and their discovery prompted DNA recombination technology The birth of it has greatly promoted the development of modern molecular biology and genetic engineering, and it is an indispensable basic tool for contemporary genetic engineering research. In bacteria, restriction endonucleases and their corresponding methyltransferases constitute a restriction-modification system to protect microorganisms from foreign phages or plasmids. Restriction enzymes and methyltransferases of the same system have the same recognition sequence on DNA, but their functions a...

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Problems solved by technology

Its disadvantage is that: after the methylation protection of host cell DNA is constructed and screened through cumbersome DNA libraries, the methylation-protected strains obtained can only specifically protect DNA from the KasI restriction enzyme or its isoschizol NarI. Cutting, few can be used for the expression of other restriction enzymes, the scope of application is narrow

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