Site-Specific Endonuclease NspLKI Is an Isoschizomer of Endonuclease HaeIII

E. V. Zabaznaya¹, L. A. Zheleznaya², I. V. Svad'bina², and N. I. Matvienko^1*

¹Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 924-0493; E-mail: nikmatv@vega.protres.ru

²Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 135-6219

^* To whom correspondence should be addressed.

Received April 16, 1998

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Site-specific endonuclease NspLKI has been isolated and purified to functionally pure state from soil bacterium Nocardia species LK by successive chromatography on columns with phosphocellulose, HTP hydroxyapatite, and heparin-Sepharose. The isolated enzyme recognizes the 5'-GG^vCC-3' sequence on DNA and cleaves it as indicated by the arrow, i.e., it is an isoschizomer of HaeIII. The final enzyme yield is 1·10⁵ units per gram of wet biomass. The enzyme is active in the temperature range of 25-60°C with an optimum at 48-55°C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM Tris-HCl, pH 7.4, 200 µg/ml albumin, 10 mM MgCl₂, and 100 mM NaCl.

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KEY WORDS: site-specific endonuclease, isoschizomer, phosphocellulose, heparin-Sepharose, hydroxyapatite