Plasmid pRARE as a Vector for Cloning to Construct a Superproducer of the Site-Specific Nickase N.BspD6I

E. A. Rogulin^1*, T. A. Perevyazova¹, L. A. Zheleznaya¹, and N. I. Matvienko²

¹Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia; fax: (0967) 330-553; E-mail: re@vega.protres.ru

²Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia; fax: (7-095) 924-0493; E-mail: nikmatv@vega.protres.ru

^* To whom correspondence should be addressed.

Received February 18, 2004; Revision received March 22, 2004

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The gene of methylase M.SccL1I that protects DNA against hydrolysis with the nickase N.BspD6I was inserted into plasmid pRARE carrying genes of tRNA, which are rare in E. coli. The insertion of the gene sscML1I into pRARE was reasoned by incompatibility of pRARE and the plasmid carrying the gene sscML1I, because both plasmids contained the same ori-site. Upon transformation of E. coli TOP10F´ cells with both the recombinant plasmid pRARE/MSsc and the expression vector pET28b containing the nickase gene bspD6IN under the phage T7 promoter, a strain of E. coli was obtained which produced 7*10⁵ units of the nickase N.BspD6I per 1 g wet biomass, and this yield was two orders of magnitude higher than the yield of the enzyme from the strain free of pRARE/MSsc.

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KEY WORDS: site-specific nickases, restriction-modification systems, superproduction, rare codons, pRARE

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