Title page for ETD etd-09192011-174830


Document Type Master's Dissertation
Author Van Wyk, Stefan George
Email stefan.vanwyk@fabi.up.ac.za
URN etd-09192011-174830
Document Title Improving the inhibitory potency of papaya cystatin, using site-directed mutagenesis
Degree MSc
Department Plant Science
Supervisor
Advisor Name Title
Prof KJ Kunert Committee Chair
Dr BJ Vorster Committee Co-Chair
Keywords
  • oryzacystatin
  • papaya cystatin
  • site-directed mutagenesis
  • cysteine protease inhibitor
  • inhibitor engineering
Date 2011-09-09
Availability unrestricted
Abstract

Novel conserved amino acid variations of papaya cystatin (PC) were investigated by amino acid substitutions using oryzacystatin-I (OCI) as a model plant cystatin for comparison. These amino acid residues in the conserved motifs are involved in binding with cysteine proteases, these include the GG (Gly-Gly) in the N-terminal region for both OCI and PC, the (Q)QVVAG (Gln-Val-Val-Ala-Gly) motif for OCI and (Q)AVVEG (Ala-Val-Val-Glu-Gly) motif for PC in the first inhibitory loop, and the PW (Pro-Trp) motif for OCI and LW (Leu-Trp) motif for PC in the second inhibitory loop. Recombinant OCI and PC mutant proteins were expressed in Escherichia coli and were tested for altered inhibitory activity against commercial cysteine proteases (papain and cathepsin L) and extracts from Colorado potato beetle (Leptinotarsa decemlineata) larvae, from banana weevil larvae (Cosmopolites sordidus) and tobacco leaf extracts (Nicotiana benthamiana). In all tests higher amounts of PC had to be used to obtain similar inhibition levels as OCI. Changing the amino acid Q at position 52 to E in OCI in the first inhibitory loop, had lowered the Ki value of the mutant against the commercial proteases. Concurrently the same amino acid string (EQ) in PC had resulted in a significantly decreased Ki value compared to PC wild-type and other mutants. All other OCI mutants were less efficient than the wild-type OCI, whereas all PC first inhibitory loop mutants had improved inhibitory activity against protease activity with the highest improvement against the protease extracts was found for the substitution of E with A at position 55. This study has shown the importance of the three conserved motifs and that it is possible to improve the binding capacity of a plant cystatins to cysteine protease activity by amino acid substitution using site-directed mutagenesis. By mutating individual amino acid residues in the first binding loop of the relatively “weak” papaya cystatin to amino acid residues found in OCI caused a significant improvement in inhibitory potency of PC.

Copyright © 2011, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria

Please cite as follows:

Van Wyk, SG, 2011, Improving the inhibitory potency of papaya cystatin, using site-directed mutagenesis, MSc (Biotechnology) dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-09192011-174830/>

C11/9/168/hv

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