Cloning and expression of a modified oryzacystatin inhibitor gene and an investigation of its inhibitory capabilities

Doctoral Thesis


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University of Cape Town

Cysteine proteinase inhibitors have shown potential as biocontrol agents for the protection of plants against insect and pathogen attack. With the advent of protein and genetic engineering such inhibitors can now be modified in order to improve their effectiveness. Because cystatins have already been isolated from plants. they provide a good starting point for developing modifications which may improve their function as biocontrol agents. The purpose of this project, therefore, was to design a potentially improved analogue of the rice cysteine proteinase inhibitor, oryzacystatin I, through molecular modelling studies. The gene sequence for this modified protein was then synthesised and expressed for kinetic analysis and insect trial assays. A prediction of the oryzacystatin I (OC I) tertiary structure was made using Biograf software on an Evans and Sutherland workstation. This structure was based on the known structures of stefin B and chicken cystatin ho se co-ordinates are published in the Brookhaven data files. Chicken cystatin is one of the most potent inhibitors of papain in the cystatin superfamily. This is believed to be due, in part, to an increased binding of the cystatin to papain through its amino-terminal region with the residues Leu7 to Gly9 playing a particularly important role.

Bibliography: leaves 128-146.