Analysis by phage display selection and site-directed retromutagenesis of the Mustard Trypsin Inhibitor 2 reactive site

The Mustard Trypsin Inhibitor (MSI) family is a small family of plant protease inhibitors so far only found in Brassicaceae. Using a phage display selection, MTI-2 (Mustard Trypsin Inhibitor 2) mutants were detected and analysed for their biochemical characteristics. Retromutants of the selected MTI-2 proteins were constructed and expressed in the Pichia pastoris system. The recombinant proteins were analysed by activity assays against bovine trypsin and Helicoverpa zea trypsin, and by circular dichroism. These analyses suggest a strict requirement for a specific proline residue adjacent to the inhibitor reactive site and give additional insights for future phage display application.