An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system’

Paul, Bindu D and Ramesh, Vaidyanathan and Nagaraja, Valakunja (1997) An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system’. In: Gene, 190 (1). pp. 11-15.

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Abstract

We had earlier overproduced the transcription activator protein C of bacteriophage Mu in a phage-T7 expression system. Although we achieved a high level of overproduction, the expression was not consistent. This could be due to the leaky expression of T7 RNA polymerase in the uninduced state. Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted in consistent, but extremely low production of the C protein. To overcome this problem, we have devised an artificial regulatory circuit to obtain stabilised, consistent overproduction of C protein. The C-binding site was cloned downstream from the transcription start point of T7 lys. Upon induction, the C protein produced binds to its site with a very high affinity, possibly acting as a transcriptional roadblock for lys. This would overcome the inhibitory effect of T7 lysozyme on T7 RNA polymerase.

Item Type:	Journal Article
Publication:	Gene
Publisher:	Elsevier
Keywords:	Bacteriophage Mu;T7 lysozyme;T7 RNA polymerase;C protein; Regulation
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	14 Jul 2004
Last Modified:	19 Sep 2010 04:13
URI:	http://eprints.iisc.ac.in/id/eprint/1003

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