METHOD OF PREPARATION OF RESTRICTION ENDONUCLEASE ECO RII FROM THE STRAIN OF BACTERIUM ESCHERICHIA COLI B 834/PSK 323 Russian patent published in 1994 - IPC

FIELD: genetic engineering, biotechnology. SUBSTANCE: biomass of strain Escherichia coli B 834/pSK 323 is suspended in potassium-phosphate buffer, pH 7.0, and disintegrating mixture is added: N-acetylmuramideglycanohydrolase and alkylphenylpolyethylene glycol at the weight ratio 0.9-1.1:190-210. After cell disrupture by ultrasonic oscillation enzyme is precipitated with polyethyleneimine adding its up to the concentration 0.3%, resuspended in potassium-phosphate buffer, pH 7.0, containing 0.4M KCl, and precipitated with with ammonium sulfate. The following separation of methylases and restriction endonucleases is carried out on DEAE-cellulose at the rate of loading and elution of enzyme 25-35 ml/g and at total eluent volume 9-11 column bed. The further purification on the phosphocellulose P-11 is carried out at the rate 25-35 ml/h. Enzyme is concentrated by dialysis against the working buffer for 5-7 h. EFFECT: enhanced degree of enzyme purification from nonspecific nucleases and increased yield of enzyme. 1 tbl