Abstract
BackgroundThe 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.ResultsWe verified that difficulties in transforming the commonly used BL21(λDE3) 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(λDE3) strain.ConclusionUsing 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.
Highlights
The pIVEX plasmids are vectors optimized for expression in the Rapid Translation System (RTS) cell-free system under control of bacteriophage T7 transcription elements
Since expression from pIVEX is not repressed by LacI, they do not contain the corresponding gene
First attempts to transform, by a standard chemical procedure, the BL21(λDE3) strain with different pIVEX failed since no transformant was obtained on LB ampicillin-agar plates
Summary
The pIVEX plasmids are vectors optimized for expression in the Rapid Translation System (RTS) cell-free system under control of bacteriophage T7 transcription elements. The continuous supply of consumable substrates and removal of reaction products provide a yield of several milligrams of protein This system uses bacteriophage T7 RNA polymerase to perform transcription, while an enriched E. coli S30 extract provides the translational machinery. Protein production in RTS requires a preliminary cloning step of the target gene into a vector, downstream of the T7 promoter For this purpose, the pIVEX family of expression plasmids has been optimized for in vitro use. The pIVEX family of expression plasmids has been optimized for in vitro use They include a T7 promoter comprising the T7 gene 10 translation enhancer [3], an efficient prokaryotic Shine-Dalgarno sequence with an optimum distance to the start codon, a multiple cloning site, and a T7 terminator which prevents 3'-terminal exonucleolytic degradation of the mRNA. These vectors are very convenient since multiple cloning sites were designed to allow gene fusions with several tags either at the N- or C-terminal of target proteins by conserving the same restriction strategy and reading frame compatibility
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