Rapid isolation of highly active RNA polymerase from Escherichia coli and its subunits by matrix-bound heparin.
1. RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin-substituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt. The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield. The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor sigma as indicated by dodecylsulfate gel electrophoresis. 2. Based on the differential affinity of the subunits of the enzyme for the heparin-carrying gel matrix, a method for separation of alpha, beta' + beta and sigma subunits by application of urea and salt-containing buffers is described. Upon recombination and dialysis with urea-free buffer 40-50% of the enzyme activity is restored.
D002846:Chromatography, Affinity / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology*; D006493:Heparin; D046911:Macromolecular Substances
D046911:Macromolecular Substances; D006493:Heparin; D012321:DNA-Directed RNA Polymerases