Preparing Plasmid Constructs to Investigate the Characteristics of Thiol Reductase and Flavin Reductase With Regard to Solubilizing Insoluble Proteinase Inhibitor 2 in Bacterial Protein Overexpression Systems

Abstract

Some proteins produced in bacterial expression systems can be insoluble and aggregated making them difficult to study. One possible solution to solubilizing such insoluble proteins is to create a fusion protein containing the protein of interest coupled with a thioreductase protein. (Yasukawa et al. J. Biol. Chem. 270, 25328-25331 (1995)) In this study we intended to assess whether thiol reductase or flavin reductase would both be equally effective at solubilizing the protein of interest; proteinase inhibitor 2. We attempted to create fusion protein constructs using the Novagen plasmid pET32a as a cloning vector since the pET32a contains thiol reductase. To compare reductase proteins, a flavin reductase gene was obtained for cloning. Due to shortage of time for this experiment, the constructs were not produced, however all of the fragments to be cloned and the control vector were produced. Additional time and resources would ensure that the constructs are produced and protein expression and analysis can be carried out to test the solubilizing properties of thiol and flavin reductases on proteinase inhibitor 2.