Site-specific endonuclease NspLKI is an isoschizomer of endonuclease HaeIII

Site-specific endonuclease NspLKI has been isolated and purified to functio nally pure state from soil bacterium Nocardia species LK by successive chro matography on columns with phosphocellulose, HTP hydroxyapatite, and hepari n-Sepharose. The isolated enzyme recognizes the 5'-GG down arrow CC-3' sequ ence on DNA and cleaves it as indicated by the arrow, i.e., it is an isosch izomer of HaeIII. The final enzyme yield is 1.10(5) units per gram of wet b iomass. The enzyme is active in the temperature range of 25-60 degrees C wi th an optimum at 48-55 degrees C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM T ris-HCl, pH 7.4, 200 mu g/ml albumin, 10 mM MgCl2, and 100 mM NaCl.