Heterologous expression and physicochemical characteristics identification of Kunitz protease inhibitor in Brassica napus

A Kunitz protease inhibitor gene (RTI; rti) was cloned from rapeseed and expressed in a Pichia pastoris expression system for the first time. After isolation and purification, the physical and chemical characteristics of the inhibitor were analyzed. The results showed that the induced expression level of the recombinant RTI reached 628 mg/L, and the specific activity of the inhibitor reached 69.6 TIU/mg protein at the shake flask fermentation level; the recombinant RTI retained more than 70% inhibitory activity between 30 and 90 °C and more than 80% inhibitory activity between pH 2.0–11.0. The metal ions Cu and CO and the organic reagents methanol, ethanol, acetone, and chloroform inhibit its activity. The recombinant RTI interacts with trypsin in a noncompetitive manner and has a strong and specific inhibitory effect on trypsin, a typical Kunitz trypsin inhibitor from plants. Combined with its good physical and chemical properties, recombinant RTI has the potential to be developed into an insect resistance protein.