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.
References
Jun 1, 1978·Journal of Bacteriology·A C Chang, S N Cohen
Aug 1, 1992·Virus Genes·T WilkV Bosch
May 5, 1991·Journal of Molecular Biology·J W Dubendorff, F W Studier
May 5, 1991·Journal of Molecular Biology·F W Studier
Jan 1, 1990·Methods in Enzymology·F W StudierJ W Dubendorff
Jul 16, 1990·Gene·D J O'MahonyS Qi
Sep 14, 1990·Gene·T L DengM D Tsai
Apr 27, 1989·Nature·W R Brown
Nov 30, 1989·Gene·J DavisonF Brunel
Jan 1, 1985·Gene·S HattmanM M Howe
Sep 7, 1988·Gene·J J DunnF W Studier
Nov 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·T R FuerstB Moss
Dec 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·Y Flashner, J D Gralla
Feb 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·S Tabor, C C Richardson
Jun 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·U DeuschleH Bujard
May 5, 1986·Journal of Molecular Biology·F W Studier, B A Moffatt
Apr 24, 1987·Cell·B A Moffatt, F W Studier
Oct 17, 1970·Nature·M ChamberlinL Waskell
Aug 15, 1970·Nature·U K Laemmli
Aug 1, 1994·Protein Engineering·V RameshV Nagaraja
May 1, 1994·Applied Biochemistry and Biotechnology·S M DeyevO L Polanovsky
Aug 1, 1994·Protein Expression and Purification·V RameshV Nagaraja
Jun 11, 1994·Nucleic Acids Research·X ChenT E Wagner
Apr 26, 1994·Proceedings of the National Academy of Sciences of the United States of America·X ChengF W Studier
Feb 1, 1995·Bio/technology·N MertensW Fiers
Citations
Nov 18, 2000·Gene·V SpehrT F Meyer
Sep 25, 2001·Blood·C C Gregorio-KingM A Kirkland
Jun 17, 1998·Biological Chemistry·S RamchandaniM Szyf
May 17, 2017·Plant Biotechnology Journal·Almudena Trapero-MozosMark A Taylor