1Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 924-0493; E-mail: email@example.com
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
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'-GGvCC-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.
KEY WORDS: site-specific endonuclease, isoschizomer, phosphocellulose, heparin-Sepharose, hydroxyapatite