AN ARTIFICIAL REGULATORY CIRCUIT FOR STABLE EXPRESSION OF DNA-BINDINGPROTEINS IN A T7 EXPRESSION SYSTEM

We had earlier overproduced the transcription activator protein C of b acteriophage Mu in a phage-T7 expression system. Although we achieved a high level of overproduction, the expression was not consistent. Thi s could be due to the leaky expression of T7 RNA polymerase in the uni nduced state. Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted in consistent, but e xtremely low production of the C protein. To overcome this problem, we have devised an artificial regulatory circuit to obtain stabilised, c onsistent overproduction of C protein. The C-binding site was cloned d ownstream 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 ove rcome the inhibitory effect of T7 lysozyme on T7 RNA polymerase.