Expression and purification of the recombinant mustard trypsin inhibitor 2 (MTI2) in Escherichia coli

The aim of this study was to define a specific procedure for the production and purification of the mustard trypsin inhibitor 2 (MTI2) using a prokaryotic system. MTI2 is a serine proteinase inhibitor isolated from ripe seeds of Sinapis alba. This protein belongs to the plant inhibitors (PIs) groups and it acts increasing plant defences against the proteolytic enzymes of many insects (especially Lepidopteran). In recent years, the recombinant MTI2 has been produced using the yeast Pichia pastoris. Here we propose an alternative and efficient procedure for the production of this protein. In particular, the synthetic mti2 gene was cloned into the pET9a vector and overexpressed in Escherichia coli. Different parameters were studied in order to optimise the yield and recovery of the recombinant protein from the insoluble fraction of protein extracts. After solubilisation with denaturants, the refolding of the protein was performed by dialysis. The renaturated, biologically active, MTI2 protein was then purified by ion-exchange and gel filtration chromatography techniques. The inhibitory activity of recombinant MTI2 was finally determined against trypsin.