Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli

Roy, Sudipta and Purnapatre, Kedar and Handa, Priya and Boyanapalli, Madanamohan and Varshney, Umesh (1998) Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli. In: Protein Expression and Purification, 13 (2). pp. 155-162.

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Abstract

We have designed a novel coupled transcriptional construct wherein Escherichia coil uracil DNA glycosylase (UDC:) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (P-trc). Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo. The system has also been exploited for purification of large amounts of Ugi. While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide cor responding to UDG. We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins. Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed.

Item Type:	Journal Article
Publication:	Protein Expression and Purification
Publisher:	Elsevier Science
Additional Information:	Copyright of this article belongs to Elsevier Science.
Keywords:	coupled transcription;bicistronic RNA;termination codon;Ung.
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	16 Dec 2009 11:19
Last Modified:	19 Sep 2010 05:28
URI:	http://eprints.iisc.ac.in/id/eprint/19438

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