Overproduction and purification of sigma 32, the Escherichia coli heat shock transcription factor.
This paper reports the overproduction and the details of a rapid method to purify active sigma 32 that is free of core RNA polymerase enzyme. Maximal overproduction of sigma 32 in a T7 RNA polymerase-based expression system is achieved only in the presence of rifampicin. This 2-day procedure involves solubilizing inclusion bodies in Sarkosyl, removal of Sarkosyl by dialysis, and a single S-Sepharose column chromatography step. The final yield of sigma 32 is about 4.1 mg of approximately 95% purity from 1 g of wet weight cells.
Nguyen
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LH
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706.
Jensen
D B
DB
Burgess
R R
RR
D002847:Chromatography, Agarose; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012293:Rifampin / Q000494:pharmacology; D012808:Sigma Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors; D014158:Transcription, Genetic
D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; D012293:Rifampin