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Title: Exploring combinatorial approaches to study protein tyrosine phosphatases
Author: Cheung, Y. W.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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The screening of support-bound libraries is a relatively new area, and has the potential to speed up the acquisition of useful data for the study of biological systems. Exploring this concept for the substrate mapping of protein tyrosine phosphatases (PTPs) was one of the goals of this thesis. The main challenges involved in such a project were, i) development of a reliable method to prepare a library of pY peptides, ii) evaluating the range of a solid supports on which to prepare and screen the libraries, and iii) designing a screen which would allow the peptides of interest to be identified. pY peptide libraries were synthesised on amino-Kieselguhr resin, using Fmoc chemistry. Kieselguhr was the resin chosen for this work as it allowed free penetration of the enzymes used, throughout the resin particles. The substrate mapping studies employed a double enzyme strategy where the products from the (first) PTP dephosphorylation reaction (i.e. phosphotyrosyl peptides), were selectively cleaved by protease, CT, (in the presence of other, phosphotyrosyl peptides). These cleaved sequences were then labelled with a dye, and the beads to which they were attached, removed for sequencing. This strategy was successfully employed to investigate the substrate specificity of leukocyte antigen related (LAR) PTP, by screening this PTP against a library of pY peptides based on residues 988-998 of the epidermal growth factor receptor autophosphorylation site (EGFR988-998), DADEpYLIPQQG. The three acidic residues (D, D and E) were replaced by one of eight amino acids. Sequencing of 6 of the "hits" from this screen showed a preference for acidic residues at all three positions, with the strongest preference at the first (D) position. Preliminary work in the exploration of a screen for PTP inhibitors was also carried out. A quenched fluorogenic tyrosyl peptide was synthesised and shown to be a substrate for CT.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available