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• W138994077 abstract "The CAP protein superfamily comprises: the Cysteine-rich secretory proteins, Antigen 5 and the Pathogenesis Related group 1 proteins. Their diverse biological functions include mammalian reproduction (e.g. CRISP1-4) and ion channel regulatory activity (e.g. Natrin, Stecrisp), plant immune responses (P14a) and the processing of conotoxins (Tex31). CAP proteins have also been identified in venoms (Triflin, Vesv5) and cancer (GLIPR1). The CAP proteins are thought to share common functions based on their highly conserved sequence motifs (namely CAP1-4) and disulfide bonds in the core of most CAP proteins. The spatial arrangement of amino acids within the large cleft of the CAP proteins suggests a possible enzymatic active site. Tex31, a cysteine-rich protein from the venom of Conus textile, was shown to be a substrate specific protease and is the only CAP protein identified with any enzymatic function. Tex31 is a multiple domains protein and its conserved CAP domain has been hypothesized to possess the catalytic activity. Over a hundred of CAP proteins have been identified but only six of them were successfully expressed using heterologous systems. Of these six, just one protein GAPR1 containing the CAP domain was solubly expressed in Escherichia coli and had its crystal structure determined. It has been difficult to express soluble CAP proteins heterologously. Therefore, this thesis aimed to (i) develop the expression protocol for Tex31 using GLIPR1 as the trial protein in Escherichia coli and Saccharomyces cerevisiae systems, (ii) to solubly express the recombinant Tex31 in E. coli or S. cerevisiae using the developed protocol in (i) and to produce enough correctly folded protein for Tex31 X-ray crystallographic analysis and protease activity assay, and (iii) to investigate the function of the CAP domain of Pry3p from S. cerevisiae using confocal microscopy.The GLIPR1 open reading frame was cloned into the pQE30 (N-terminal 6× His-tag) and pQE60 (C-terminal 6× His-tag) vectors in the E. coli strain M15[pREP4] and into the PDR5 locus of S. cerevisiae. The in frame and correct DNA sequence of GLIPR1 in pQE vectors and PDR5 locus were confirmed by DNA sequencing. The expression of GLIPR1 was not detected using SDS-PAGE gel and western blot analysis. Disruption in the T5 promoter region of pQE30 vector was identified by DNA sequencing but it remained unknown as to why GLIPR1 protein did not express in pQE60/E. coli and S. cerevisiae systems. Nevertheless, the main aim of this section which was to develop the expression protocol for Tex31 had been achieved.The E. coli expression protocol developed for GLIPR1 in (i) was applied to Tex31. Multiple constructs of Tex31 were cloned into pQE30 and pQE60 systems. Initial expression of Tex31 resulted in the formation of inclusion bodies. Refolding experiments were attempted but the refolded Tex31 was unsuccessful due to Tex31 instability. Similar Tex31 constructs were expressed in E. coli strain DH5α as soluble protein using a maltose-binding protein (MBP) as a fusion…" @default.
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• W138994077 date "2012-01-01" @default.
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• W138994077 title "Investigation of CAP proteins: GLI pathogenesis-related protein 1, Tex31 and Pry3p" @default.
• W138994077 hasPublicationYear "2012" @default.
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