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

Bindu Paul, Vaidyanathan Ramesh, Valakunja Nagaraja

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)11-15
Number of pages5
JournalGene
Volume190
Issue number1
DOIs
Publication statusPublished - Apr 29 1997
Externally publishedYes

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Keywords

  • Bacteriophage Mu
  • C protein
  • Regulation
  • T7 lysozyme
  • T7 RNA polymerase

ASJC Scopus subject areas

  • Genetics

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