An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system.
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.
Paul
B D
BD
Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
Ramesh
V
V
Nagaraja
V
V
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D009113:Muramidase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011486:Protein C / Q000378:metabolism; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D011486:Protein C; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009113:Muramidase