Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Chromatography, Agarose
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Escherichia coli / genetics*
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Gene Expression
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Genetic Vectors
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Heat-Shock Proteins / biosynthesis*
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Heat-Shock Proteins / genetics
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Heat-Shock Proteins / isolation & purification
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Recombinant Fusion Proteins / biosynthesis*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Rifampin / pharmacology
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Sigma Factor / biosynthesis*
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Sigma Factor / genetics
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Sigma Factor / isolation & purification
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Transcription Factors*
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Transcription, Genetic
Substances
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Heat-Shock Proteins
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Recombinant Fusion Proteins
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Sigma Factor
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Transcription Factors
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heat-shock sigma factor 32
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Rifampin