Generation of Recombinant Plasmid Constructs to Assess the Ability of NADH:Flavin Oxidoreductase to Solubilize Proteinase Inhibitor 2 in Bacterial Protein Overexpression Systems

Abstract

A possible solution to the problem of conversion of overexpressed proteins into insoluble aggregates in bacterial protein expression systems is to express the target protein fused to a reductase enzyme. One such example is Escherichia coli thiol reductase (Trx), currently used in the Novagen pET expression system marketed by EMD Biosciences. In the present study, an alternative redox protein, the highly soluble E.coli NADH:flavin oxidoreductase (Fre) was chosen to investigate whether it would solubilize a protein called proteinase inhibitor 2 (PI2), which is relatively insoluble when expressed in high concentration. In order to compare the properties and characteristics of thiol reductase and flavin oxidoreductase, attempts were made to generate fusion constructs of trx:PI2 and fre:PI2, as well as a control construct with PI2 by itself, using the Novagen pET32a vector plasmid. + + Due to time constraints and a problem with amplification of fre, the pET32a Δ trx / fre /PI2 + construct was not generated. However, potential clones of the pET32a/PI2 and + pET32a Δ trx /PI2 recombinant constructs were generated and isolated. Further study is + + necessary to confirm the identity of these constructs, to generate the pET32a Δ trx / fre /PI2 construct, and to express these constructs under appropriate conditions to assess and compare the effects of flavin oxidoreductase and thiol reductase on solubility of PI2.