A system for heterologous expression and isolation of Escherichia coli RNA polymerase and its components

A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein- and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits alpha, beta or be...

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Bibliographic Details
Published in:Biochemistry (Moscow) 2007-02, Vol.72 (2), p.178-187
Main Authors: Khodak, Yu A, Koroleva, O N, Drutsa, V L
Format: Article
Language:English
Subjects:
Base Sequence
Binding Sites
Chitin
DNA-Directed RNA Polymerases - isolation & purification
DNA-Directed RNA Polymerases - metabolism
E coli
Enzymes
Escherichia coli
Escherichia coli - enzymology
Escherichia coli Proteins - isolation & purification
Escherichia coli Proteins - metabolism
Genetic Vectors - genetics
Genetic Vectors - metabolism
Inteins
Molecular Sequence Data
RNA polymerase
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Summary:A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein- and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits alpha, beta or beta' in conjunction with the natural subunits alpha, beta, beta', and sigma can participate in RNA polymerase assembly in vivo, providing affinity-based isolation of the enzyme. Functional activity of the enzyme preparations was demonstrated in the experiments on in vitro transcription and promoter complex formation. With the use of IMPACT technology, sigma(70) subunit can be isolated as an individual protein without admixture of RNA polymerase.
ISSN:0006-2979
1608-3040
0320-9725
DOI:10.1134/S0006297907020071