Purification of Eco R1 endonuclease on heparin Sepharose-4B column chromatography

Abstract

Restriction endonucleases play a very important role in genetic engineering and DNA mapping. Among hundreds of restriction endonucleases, the Eco R1 enzyme is the most useful and widely investigated enzyme. After sonication and ultracentrifugation, crude extracts of E. coli RY 13 were purified by employing the polyethyleneimine precipitate, ammonium sulfate precipitate and heparin Sepharose-4B affinity column chromatography. The Eco R1 enzyme were purified at about 42 folds and the specific activity was about 100,000 U/mg of protein. The whole purification procedure was finished within two days. The recovery was about 42%. The enzyme was sufficiently concentrated for direct specific DNA hydrolysis.