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 40-160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine beta-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.
Subject(s)
Mustard Plant/metabolism , Plant Proteins/pharmacology , Plants, Medicinal , Trypsin Inhibitors/chemistry , Trypsin/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , Fermentation , Kinetics , Molecular Sequence Data , Mustard Plant/genetics , Pichia/growth & development , Plant Proteins/chemistry , Plant Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Trypsin Inhibitors/genetics , Trypsin Inhibitors/pharmacologyABSTRACT
The gene coding for the mustard trypsin inhibitor-2 has been isolated from a genomic library and characterized. Comparison of genomic and cDNA sequences indicates that the gene is interrupted by an intron of 193 bp. The eukaryotic peculiar regulatory sequences have been detected in the 5' flanking region of the gene. In addition, a decanucleotide has been detected that is highly similar to the proposed G-box and to the ABRE motifs required for the gene expression induced by methyl jasmonate and abscissic acid. Northern blot analysis demonstrates that the gene is expressed in immature seeds as well as in wounded leaves.