Subcloning emoA into pET32a(+), pET32a(+)ΔtrxA/+fre and pET32a(+)ΔtrxA to Assess the Relative Effect of Thioredoxin A and Flavin Oxidoreductase on emoA Solubility

Abstract

The Novagen pET32a(+) system is widely used for high-level expression of recombinant proteins. Key to its success is the thioredoxin (TrxA) protein that allows the peptide product to stay in a soluble state. Flavin oxidoreductase ( fre ) is another oxidoreductase which could be used to increase protein solubility of highly expressed peptides. To investigate the solubility-enhancing effects of fre in comparison to trxA, EDTA monooxygenase A ( emoA ), a relatively insoluble protein, was incorporated into the original pET32a(+) vector as well as a genetically modified pET32a(+) vector with fre as the replacement for trxA. The new constructs were attempted by isolation of emoA by gel purification, T4 DNA ligation to insert the PCR product into the vector and calcium chloride transformation of the new constructs into DH5 α E. coli. None of the isolated transformants were shown to be the desired vectors in the verification stage after transformation. As such, this report troubleshoots the problems faced and guides future experiments to get the appropriate clones.