A New Method for the Large Scale Purification of Escherichia coli Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

Abstract

This paper describes a method for the puritication of Escherichia coli DNA-dependent RNA polymerase which is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 3 kg of cells.The method involves disruption of the bacterial cells with glass beads in a Waring Blendor, digestion with DNase I, centrifugation to remove ribosomes and debris, fractionation with ammonium sulfate, and chromatography on diethylaminoethyl cellulose, phosphocellulose, and Bio-Gel A-1Sm.Addition of 5% glycerol and 0.1 mu dithiothreitol to all buffers substantially increases the stability of the enzyme throughout the purification.Depending on how cells are grown, 100 to 300 mg of polymerase are obtained from 1 kg of cells.The polymerase is greater than 98% pure and free of contaminating nucleases.