Investigation of MBP tag removal from recombinant potato type II proteinase inhibitor and testing of serine protease inhibition

Abstract

Potato type II proteinase inhibitor (PI2) is a small, disulfide-rich serine protease inhibitor that requires careful folding to retain its functionality. Due to the promising applications of PI2 in medicine, cancer treatment and biotechnology, researchers have been interested in optimizing its large-scale production. However, as a result of the small size and complex conformation of PI2, large affinity tags such as maltose-binding protein (MBP) complicate its ability to be purified and can potentially interfere with its inhibitory function. The present study investigates whether the removal of the MBP fusion tag affects the inhibitory function of PI2. MBP-PI2 recombinant protein was expressed in E. coli SHuffle (C3028) and purified by amylose gravity affinity chromatography. A subsequent factor Xa cleavage optimization assay suggested inefficient MBP tag removal from recombinant MBP-PI2, indicating the possibility that PI2 inhibits factor Xa, and therefore MBP-tagged PI2 may be sufficiently functional. Further investigation of MBP-PI2 functionality in a trypsin inhibition assay appeared to show trypsin cleaving MBP-PI2. Subsequent bioinformatic analysis predicted potential non-specific cleavage of MBP and PI2, suggesting that trypsin is unsuitable for PI2 functional assays. Nonetheless, these results provide insight into the functionality of MBP-PI2, allowing further exploration into the therapeutic potential of the protein.