Abstract
Overexpression of recombinant proteins in Escherichia coli can result in biologically inactive aggregates of misfolded proteins known as inclusion bodies. The fusion of thioredoxin A (TrxA) to target proteins has been used in commercial plasmids to increase the solubility of overexpressed protein which can reduce the formation of inclusion bodies. The objective of this study was to create recombinant plasmid vectors that would overexpress TrxA fused to proteinase inhibitor II (PI2), a protein domain containing 8 disulphide bonds known that is known to form inclusion bodies. We hypothesized that the co-expression of TrxA in either cis or trans with PI2 would increase levels of soluble PI2 when overexpressed in E. coli. We designed oligonucleotide primers to amplify the PI2 gene from plasmid pE32PI2 using polymerase chain reaction. We found that the addition of dimethyl sulfoxide at concentrations between 0.25% and 1.0 % was required for PCR amplification of the PI2 gene from pE32PI2. In this study the PI2 gene was successfully amplified by PCR using DMSO as an additive. The expected DNA band sizes were confirmed by gel electrophoresis.