Construction of Recombinant Expression Vectors to Study the Effect of Thioredoxin on Heterologous Protein Solubility

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. SDS-PAGE analysis did not detect induction of the thioredoxin-PI2 fusion protein or the apo-PI2 protein, leading us to speculate that PI2 may not be a suitable protein for high level protein expression studies using the pET-32 vector series in E. coli strain BL21.