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

Abstract

The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C-terminal amino acids of the precursor form on the inhibitor activity, the C-terminal precursor and the mature protein were both expressed. A third His-tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 401 60 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine L L-trypsin compared to the precursor 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 proteases.z 2000 Federation of European Biochemical Societies.

Functional Expression on Bacteriophage of the Mustard Trypsin Inhibitor MTI-2

“…The Pichia pastoris strain GS115 and the pPIC9 plasmid were purchased from Invitrogen (San Diego), as part of the Pichia expression kit. Media have been described in (10). Bovine pancreatic ␤ trypsin type III (EC 3.4.21.4) and bovine pancreatic ␣ chymotrypsin type II (EC 3.4.21.1) were purchased from Sigma Chemical Co. (St. Louis).…”

“…Triplets correspond to amino acid codons; restriction sites are under-lined. Numbers above the sequences correspond to nucleotide positions in the mti-2 gene region encoding the mature protein (10 Subcloning into pRlac3 vector. The DNA coding for the mature part of MTI-2, and for variants PAI, PLI, and AAA was transferred into pRlac3 (11) where it was fused before the bacteriophage M13 gene 3.…”

“…Individual colonies were expressed in 1 ml medium, and high-expressing colonies were selected by analysis of 15 l culture supernatant on a 16.5% Tris-tricine SDS PAGE gel, and silverstaining. Selected high expressors were grown in 50 ml medium, and purified as described in (10).…”

“…The inhibition of trypsin activity by the MTI-2 mutants was measured as described by (10). For bovine ␣-chymotrypsin, the active concentration was titrated by the method of Bender (12) using cinnamoylimidazole (N-TCI), indicating that chymotrypsin was 64% active.…”

“…This latter property makes it an interesting starting molecule for engineering inhibitory properties. The natural defensive function of MTI-2 has been demonstrated by the observations that its expression is induced in wounded leaves (7), that transgenic tobacco plants expressing MTI-2 show enhanced resistance against Spodoptera littoralis (8), Plutella xylostella and Mamestra brassicae (9), and that it inhibits S. exigua gut proteinases in vitro (10).…”