Purification, Properties and Recognition Sequence of Site-specific Restriction Endonuclease from Acetobacter liquefaciens AJ 2881

Abstract

A type II restriction endonuclease, designated as AliAJI, was purified from cells of Acetobacter liquefaciens AJ 2881 by combined column chromatography on heparin-Sepharose CL-6B, DEAE-Sepharose CL-6B and blue Sepharose CL-6B. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis, and the enzyme preparation was free from other nuclease activities, as judged by constancy of lambda DNA-digest electrophoretic patterns after prolonged incubation for 24hr. The enzyme was optimally active at 37°C at pH7.5, required neither sodium chloride nor ammonium sulfate, both of which rather inhibited enzyme activity at high concentration (100 and 75mM, respectively), and cleaved lambda, φX174 RF, SV40, pBR322, M13 mp7 RF and Ad2 DNAs at 18, 1, 2, 1, 1 and 25 or more sites, respectively. The recognition sequence of the enzyme on DNA molecules was determined to be 5'-C-T-G-C-A-G-3', and the enzyme was found to cut between A and G in the sequence, being an isoschizomer of the endonuclease of Providencia stuartii 164 (PstI).