A System for Heterologous Expression and Isolation of Escherichia coli RNA Polymerase and Its Components

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Yu. A. Khodak¹, O. N. Koroleva², and V. L. Drutsa^1*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: drutsa@genebee.msu.su; hod@freemail.ru

²Chemical Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: koroleva@genebee.msu.su

^* To whom correspondence should be addressed.

Received September 19, 2006; Revision received October 16, 2006

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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, sigma⁷⁰ subunit can be isolated as an individual protein without admixture of RNA polymerase.

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KEY WORDS: E. coli RNA polymerase, subunits, affinity chromatography, IMPACT-system, transcription

DOI: 10.1134/S0006297907020071

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