Use of pIVEX plasmids for protein overproduction in Escherichia coli -: art. no. 18

Background: The pIVEX plasmids are vectors optimized for expression in the Rapid Translation System (RTS) cell-free system under control of bacteriophage T7 transcription elements. Even if these plasmids are intended for use in vitro, it is usually worthwhile to compare both cell-free and bacterial expression from the same genetic construct. However, some RTS users encountered problems when they introcuded these plasmids into Escherichia coli host strains producing the T7 RNA polymerase. Results: We verified that difficulties in transforming the commonly used BL21(lambda DE3) strain with pIVEX arose from the presence of a strong T7 promoter combined with a high-copy number plasmid, independent of gene expression. When these vectors were introduced into this strain harboring a compatible plasmid carrying the lactose repressor ( lacI), we improved the transformation efficiency by 4 orders of magnitude. Moreover, we designed a transformation protocol that allows, after induction, the overproduction of pIVEX-encoded proteins in the BL21(lambda DE3) strain. Conclusion: Using the correct plasmid/host combination and transformation-expression protocol, we could directly compare overproduction of the same pIVEX-encoded proteins from both in vivo and in vitro expression systems.