Expression, purification, and functional characterization of potato proteinase inhibitor type II (PI2)

Abstract

Proteinase inhibitors regulate the activity of proteinases, which catalyze the breakdown of other proteins. They are commonly found in plant species to confer protection against external stressors but may also play a role in human health. As such, researchers have aimed to optimize the large-scale recombinant production of proteinase inhibitors with potential clinical applications. One example is potato proteinase inhibitor type II (PI2), which inhibits the activity of serine proteases such as trypsin and has anticarcinogenic and appetite suppressing properties. PI2 requires diligent formation of its multiple disulfide bonds to maintain its functionality. As such, previous studies have used SHuffle Escherichia coli strain, which has a favorable cytosolic environment for disulfide bond formation, to produce maltose-binding protein tagged PI2 (MBP-PI2). The present study investigates whether PI2 expressed in SHuffle E. coli cells is functional by developing an assay to evaluate its trypsin inhibition activity. Results from this assay revealed that MBP-PI2 produced by SHuffle cells may be capable of inhibiting trypsin activity and that this functionality may be conferred by PI2 rather than the attached MBP tag. Thus, SHuffle E. coli strain may be a suitable expression host for large-scale production of functional PI2 for clinical applications. Additionally, the developed trypsin inhibition assay may be adapted for exploration into functionality of other serine protease inhibitors.