Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase.
A method for the purification of highly pure and active Escherichia coli RNA polymerase holoenzyme is described. This method is simple, reproducible, and can be performed at room temperature. The procedure involves the high-performance liquid chromatography of a partially purified RNA polymerase sample on a Mono Q ion-exchange column. Under the conditions used, RNA polymerase holoenzyme is well separated from the core RNA polymerase and other impurities. The purified RNA polymerase contains virtually no impurities as judged by SDS-polyacrylamide gel electrophoresis. The purified RNA polymerase holoenzyme contains the sigma 70 subunit in stoichiometric amounts and is at least 90% active.
Hager
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McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Jin
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Burgess
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D000837:Anion Exchange Resins; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D012117:Resins, Synthetic
D000837:Anion Exchange Resins; D012117:Resins, Synthetic; C042855:Mono Q; D012321:DNA-Directed RNA Polymerases