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

摘要

The present invention relates to isolated DNA coding for the restriction endonuclease ScaI as well as to a method for cloning methylase genes from Streptomyces into E.coli by a modification of the methylase selection method. At first, the standard methylase gene selection method was tried to clone the ScaI methylase gene using a high-copy-number cloning vector pUC19 during library construction. The ScaI methylase gene was refractory to cloning by using pUC19, presumably due to the poor expression of the ScaI methylase gene in E.coli. If the ScaI methylase is not efficiently expressed in E.coli, the ScaI sites on the plasmid will not be sufficiently modified by the methylase. As a consequence, the plasmid will be cleaved and lost in the plasmid library after ScaI endonuclease challenge. Since the standard methylase selection did not work, the "endo-blue method" was tried to clone the ScaI endonuclease gene. Nineteen blue colonies were identified, but none of them yielded any detectable ScaI endonuclease activity. The ScaI endonuclease gene was first cloned by inverse PCR using primers that annealed to the end of the ScaI methylase gene. In order to increase the ScaI endonuclease expression in E. coli , an optimal ribosome binding site and spacing were engineered in front of the ATG start codon and the gene was inserted into expression vector pRRS.