Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris

The mustard trypsin inhibitor MT12 was expressed as secretory protein in th e yeast Pichia pastoris. In order to evaluate the influence of the C-termin al amino acids of the precursor form on the inhibitor activity, the C-termi nal precursor and the mature protein were both expressed. A third His-tagge d construct was also designed to compare alternative purification procedure s. Proteins were efficiently expressed at levels of 40-160 mg/l in shake fl asks. Equilibrium dissociation constants demonstrated that the mature prote in was a stronger inhibitor of bovine beta-trypsin compared to the precurso r and His-tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut prote ases. (C) 2000 Federation of European Biochemical Societies.