Overproduction and purification of the omega subunit of Escherichia coli RNA polymerase.
This paper reports the construction of plasmids which direct the overproduction of the omega subunit of Escherichia coli RNA polymerase and the subsequent purification of omega. Useful overproduction is achieved only if the natural ribosomal binding site region of rpoZ is replaced with the ribosomal binding site region of bacteriophage T7 gene 10. Overproduction is directed by T7 RNA polymerase which is provided on a separate plasmid. omega is purified by three column steps either from the insoluble inclusion body fraction or from the soluble fractions of lysates. The final yield is approximately 2 mg omega per 10 g cells wet wt. Additionally, we found that recombinant omega is readily cleaved by an endogenous protease. Sequence analysis of the most prevalent proteolytic fragment suggested that the protease responsible was the product of the ompT gene. Cleavage of omega is greatly reduced in ompT- strains.
Gentry
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McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706.
Burgess
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases