Attempted Construction of a pET32a(+) Vector Containing EDTA Monooxygenase A

Abstract

Protein overexpression vectors, such as Novagen’s pET vectors are very efficient cloning and expression systems for recombinant proteins in Escherichia coli. It has been shown that overexpression of heterologous proteins can lead to their aggregation within the cell known as inclusion bodies. Novagen’s pET32a(+) vector reduces the formation of inclusion bodies by increasing the solubility of proteins when fused directly to highly soluble thioredoxin A (TrxA). In this study, we designed PCR primers that allowed for cloning of a translational fusion between TrxA and EDTA monooxygenase (EmoA). We successfully amplified emoA from the pEmoA plasmid in an effort to subclone the gene into pET32a(+). We confirmed the necessity of DMSO as a PCR enhancer and demonstrated that concentrations ranging from 5 – 9% were adequate to amplify emoA. Putative constructs were screened by colony PCR and restriction digests in an effort to identify positive clones. This analysis established that emoA had not been inserted into pET32a(+) and further attempts to clone and identify the construct were futile.