Overproduction and purification of sigmaS, the Escherichia coli stationary phase specific sigma transcription factor.
This paper reports the overproduction and the details of a rapid method to purify active sigmaS monomers from a T7 RNA polymerase-based protein expression system. This 2-day procedure involves solubilizing inclusion bodies in sarkosyl detergent, removal of sarkosyl by dialysis, and a single gel filtration column chromatography step. The final yield of sigmaS is about 9 mg of approximately 92% purity from 0.5 g of wet weight cells. Overproduced sigmaS binds to core RNA polymerase and supports transcription from the bolAp1 promoter, a sigmaS-dependent promoter.
Nguyen
L H
LH
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.
Burgess
R R
RR
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D012808:Sigma Factor / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D012808:Sigma Factor; C025231:sarkosyl; D012521:Sarcosine