Improvement of the T7 expression system by the use of T7 lysozyme.
One of the most efficient systems for the high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulated transcription unit supplying the specific T7 RNA polymerase. Various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase are in use. Most of them show high levels of expression in non-induced cells, low factor of induction or impaired growth of host cells. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is suppressed by the use of the genes for the Lac repressor and T7 lysozyme, integrated on the expression vector. T7 lysozyme expression is probably down-regulated in the induced expression system by antisense RNA. This overcomes the inhibitory effect of T7 lysozyme on T7 RNA polymerase as shown by SDS PAGE and flow cytometry analysis of expressed GFP. The main features of the expression vector compared with other systems are low background, high factor of induction and unaffected growth of non-induced cells.
Spehr
V
V
Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstr. 21/22, 10117, Berlin, Germany.
Frahm
D
D
Meyer
T F
TF
D017123:Bacteriophage T7 / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009113:Muramidase / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009113:Muramidase