Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3).
The T7 polymerase-based pET System is one of the most powerful and widely used prokaryotic expression systems available today. Expression of even slightly toxic gene products in BL21 (DE3), however, has been problematic due to basal expression, which leads to decreased plasmid stability and variable yields following large-scale growth and induction. Use of host strains such as BL21 (DE3) pLysS provides high stringency and consistent expression but typically at the cost of reduced protein levels upon induction. The experiments presented here suggest that catabolite repression can effectively reduce basal expression of the T7 polymerase gene in BL21 (DE3), yielding tight regulation and consistency comparable to that of BL21 (DE3) pLysS. By switching to a poor carbon source for the final growth cycles, the higher expression levels typical of BL21 (DE3) can readily be obtained upon induction.
Pan
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Schering-Plough Research Institute, 2015 Galloping Hills Road, Kenilworth, NJ 07033, USA.
Malcolm
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D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000821:virology; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007763:Lac Operon; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase