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Screening for extracellular protein - protein interactions in a novel yeast growth selection system

Urech, David M.. Screening for extracellular protein - protein interactions in a novel yeast growth selection system. 2004, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7384

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Abstract

The recent sequencing of entire eukaryotic genomes revealed a great number of open reading frames (ORFs), most of which potentially code for proteins of unknown function. Identifying interaction partners may facilitate the functional characterisation of unknown gene products. Moreover, large-scale approaches to identify protein interactions may be used to untangle the complete interaction network of all the gene products expressed in a given organism, the so-called interactome. Currently, the yeast two-hybrid system is the most widely used genetic assay for large-scale detection of protein – protein interactions and it has successfully been applied to map the interactome of several organisms. However, since interactions in this system are detected in the nuclear environment, it does not account for the particular biochemical requirements of extracellular and integral membrane proteins. In the first part of this thesis, I describe a novel genetic growth selection system to detect interactions between extracellular and transmembrane proteins in a topologically extracellular compartment of Saccharomyces cerevisiae. In this system the proteins of interest are expressed as fusions to mutually complementing mutant derivatives of the yeast ER resident transmembrane receptor Ire1p. Interaction between the proteins of interest causes dimerisation of the Ire1p moieties allowing for complementation and activation of Ire1p. Active Ire1p in turn triggers a signalling cascade, which induces expression of selectable reporter genes in the nucleus and promotes growth under selective conditions. The feasibility of this system to monitor interactions between extracellular proteins was demonstrated by specific pairings of epitope and single-chain Fv (scFv) antibody fragments. In part two, I describe the application of this system in a screening procedure to select scFv antibody fragments that specifically bind to human Interleukin-13 (hIL13). In a first round, hIL-13 binders were selected from a scFv-antibody library by yeast growth under selective conditions. In order to improve their binding affinity, parts of the scFv fragments obtained from the primary screening were randomised by homologous recombination in yeast and subjected to growth selection under increased selective conditions.
Advisors:Riezman, Howard
Committee Members:Spiess, Martin
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spiess)
UniBasel Contributors:Spiess, Martin
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7384
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:106
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 15:25

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