Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system.
Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the pET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow and produce innocuous target proteins indefinitely. Different configurations of the expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products.
Studier
F W
FW
Biology Department, Brookhaven National Laboratory Upton, NY 11973.
D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*
D012321:DNA-Directed RNA Polymerases; D009113:Muramidase