Abstract
The pET-32 vector series is designed for high-level expression of recombinant thioredoxin fusion proteins. Previous studies have shown that linkage to thioredoxin increases the yield of biologically active proteins expressed in Escherichia coli. How thioredoxin promotes protein solubility is not known, although a catalytic contribution involving the formation of disulphide bonds in the E. coli cytoplasm has been proposed. In this study, we have cloned the proteinase inhibitor II (PI2) gene from potato into wild type pET-32a and previously created pET-32aΔtrx vectors. PI2 was chosen as a model protein to study the oxido-reductase activity of thioredoxin since it contains eight disulphide bonds. These new vectors allow comparison of the solubility of overexpressed PI2 fused to thioredoxin to apo-PI2. To our knowledge, this was the first attempt to express PI2 in a prokaryotic system in E. coli strain BL21. SDS-PAGE analysis did not show detectable induction of PI2 under the conditions tested. We speculate that PI2 may be unsuitable for expression under these conditions and further optimization will be required.