Browsing by Author "Maeder, Dennis"

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    Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylophic yeast Pichia pastoris
    (1996) Hüsler, Jennifer; Klump, H H; Brandt, Wolf F; Maeder, Dennis
    Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and deletions within the sequence have also been investigated. Thus, this family of inhibitors is considered useful as a model system for the study of protein-protein interactions. Curcrbita maxima trypsin inhibitor I (CMTI I) may be thought of as representative of the squash inhibitors, for which there is detailed structural and functional information available. It is a 29 amino acid protein, with the tri-disulphide bridging pattern common to all squash inhibitors. There are only a few examples of squash inhibitors being produced by recombinant DNA technology. As this technique offers a relatively cheap way of producing large amounts of these proteins, further investigation is required. Problems have been experienced with the expression of disulphide-rich proteins in E. coli, as the cytosol of this microorganism is not conducive to their folding. Furthermore extraction of these proteins from the peri plasmic space is often required, resulting in a reduction in yield. To overcome these shortcomings, the methylotrophic yeast Pichia pastoris was investigated as an alternative means of expression, although at the inception of this work, no disulphide-rich proteins of this size had been expressed in P. pastoris. It was a challenge to investigate the feasibility of producing squash inhibitors in this expression host and to compare the activity of the recombinant inhibitor to that of native CMTI I.
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    Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris
    (1996) Hüsler, Jennifer; Klump, Horst H; Brandt, Wolf F; Maeder, Dennis
    Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and deletions within the sequence have also been investigated. Thus, this family of inhibitors is considered useful as a model system for the study of protein-protein interactions. Cucurbita maxima trypsin inhibitor I (CMTI I) may be thought of as representative of the squash inhibitors, for which there is detailed structural and functional information available. It is a 29 amino acid protein, with the tri-disulphide bridging pattern common to all squash inhibitors. There are only a few examples of squash inhibitors being produced by recombinant DNA technology. As this technique offers a relatively cheap way of producing large amounts of these proteins, further investigation is required. Problems have been experienced with the expression of disulphide-rich proteins in E. coli, as the cytosol of this microorganism is not conducive to their folding. Furthermore extraction of these proteins from the peri plasmic space is often required, resulting in a reduction in yield. To overcome these shortcomings, the methylotrophic yeast Pichia pastoris was investigated as an alternative means of expression, although at the inception of this work, no disulphide-rich proteins of this size had been expressed in P. pastoris. It was a challenge to investigate the feasibility of producing squash inhibitors in this expression host and to compare the activity of the recombinant inhibitor to that of native CMTI I.
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    Cloning and expression of a chimeric protease inhibitor encoding gene in Escherichia coli and Pichia pastoris
    (1996) Matsebula, Aaron Mfanuzile; Botes, DP; Klump, HH; Maeder, Dennis
    Squash family protease inhibitors are small peptides of 27-32 residues, hence they are ideal subjects for structure-function studies. Their small size is within the reach of peptide chemical synthesis, which enables one to produce enough peptide material for experimental purposes within a reasonable time frame.
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    Cloning and expression of a modified oryzacystatin inhibitor gene and an investigation of its inhibitory capabilities
    (1997) Haworth, Caroline Joanne; Thomson, Jennifer Ann; Maeder, Dennis
    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.
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    The investigation of a novel proteinase inhibitor as a means to transfer insect resistance to plants
    (1996) Tasker, Jacqueline Ruth; Thomson, Jennifer Ann; Maeder, Dennis; Botes, Dawie
    A viable IPM programme involves a clear understanding of and the use of a number of components, the most important being the bionomics of insect pests; monitoring systems to establish the prevalence and seasonal occurrence of insect pests; calculation of economic thresholds; the biology of parasites and predators; utilisation of insect resistant plant varieties; and methods to maximise the advantages of pesticides and minimise their disadvantages. Insect-resistant crop varieties form an important part of IPM. There is usually no extra cost to the farmer once the resistant variety has been obtained and it is easily available to him thereafter. In the context of an IPM strategy, plant resistance should improve the impact on a pest population when both biological and chemical control methods are used.