Separation of Escherichia coli RNA polymerase sigma-70 holoenzyme from core enzyme on heparin-Sepharose columns.
A method is described for the rapid purification of DNA-dependent RNA polymerase sigma-70 holoenzyme from Escherichia coli. The essential step in this protocol involves the differential elution of sigma-70 holoenzyme from core polymerase on a heparin-Sepharose column. Using a linear gradient of KCl, holoenzyme was found to elute at 0.25 M whereas core polymerase eluted at 0.35 M. From 20 g of cells, up to 1 mg of RNA polymerase holoenzyme could be isolated in two days. The preparations were greater than 95% pure with respect to protein, and saturated with the sigma subunit.
Wellington
S R
SR
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Spiegelman
G B
GB
D002850:Chromatography, Gel / Q000379:methods*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D012685:Sepharose / Q000031:analogs & derivatives
C037405:heparin-sepharose; D012685:Sepharose