Production of a Recombinant Vector to Enable the Study of Thioredoxin Function as a Bound or Detached Solubilizer of Proteinase Inhibitor 2 in a Bacterial Protein Overexpression System

Abstract

A useful method of preventing formation of insoluble aggregates during bacterial protein overexpression is to fuse a reductase enzyme to the expressed protein. One example of this is the pET system from Novagen. The pET32a plasmid is an expression vector that fuses insert gene sequences to thioredoxin. This construct has been shown to reduce protein aggregates even when not bound to the protein. In this study, a vector with the fusion tag removed and a fully separate thioredoxin reinserted was created to enable expression of both proteins separately from the same plasmid. This vector is based on a preexisting plasmid with the thioredoxin tag removed and a proteinase inhibitor 2 gene inserted. The thioredoxin sequence along with the appropriate promoter and ribosome-binding site from the commercial pET32a plasmid were isolated. This difficult procedure was followed by the insertion of the sequence into the previously created vector. This study will require further work to get a conclusive result, but potential candidates were isolated.