Application of phage display to the study of toxin-receptor interactions

McLean, Hector Alexander (1999) Application of phage display to the study of toxin-receptor interactions. PhD thesis, University of Glasgow.

Abstract

In causing disease, most bacterial toxins must firstly interact specifically with one or more host receptors. Thus, discovery of the identity of these receptors is an important step towards elucidating a toxin's mode of action. This project aimed to assess whether the technique known as phage display had any role to play in the identification of toxin receptors. Libraries of DNA were supplied for this work. These libraries had degenerate DNA of defined sequence length cloned into genes encoding major or minor components of the capsid of filamentous phage of the Ff family. This resulted in random peptides being 'displayed' in the outer envelope of the phage. Libraries of such phage were exposed to surfaces of immobilised toxin, with those carrying a random peptide sequence complementary to the receptor binding site interacting with, and being bound to, the toxin. Unbound phage were washed away and bound phage were removed by elution with a specific or non-specific agent. The toxin used as a model in this work was the heat-labile toxin of enterotoxigenic Escherichia coli (LT), for which the natural receptor is already known. A diarrhoeal toxin almost identical to that from Vibrio cholerae, it recognises a glycolipid, ganglioside GM1 This assessment therefore called for the peptide extension of the phage to mimic a natural carbohydrate moiety. The structure of LT is designated AB5, signifying that it has one Active and five Binding subunits. As this work was primarily concerned with binding, isolated LT-B pentamers, secreted by a genetically-modified marine Vibrio were used in the experimental work in preference to whole toxin. After examining the growth pattern of this strain, and relating it to B subunit production, the pentamers were purified by affinity chromatography using a galactose column, then biotinylated to facilitate attachment to streptavidin-coated plastic. The biotinylated product was tested for integrity and activity. Three phage display libraries were assessed. After exposure to the toxin, the eluted phage were tested in binding assays and, where appropriate, were sequenced to determine the amino acid composition of the random insert. The first library tested, the 'Smith hexamer', carried a six amino acid extension to minor capsid protein III and performed poorly: the selected clones showed no specific binding to the toxin. The second library, the 'Luzzago nonamer', which carried a nine amino acid extension to protein VIII, was an example of a library carried on a phagemid vector. It apparently had suffered a flaw in construction and appeared unable to synthesise coat protein. The third library, the 'Smith pentadecamer', carried a fifteen amino acid extension to protein VIII. Successive rounds of exposure to the toxin and amplification of binding phage demonstrated that, although selection of phage carrying specific peptides was occurring the affinity was for elements of the test system other than LT-B itself. Thus, these experiments were unable to identify peptides from a random, phage-borne library able to mimic the natural receptor for LT-B.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Dr. Robert Aitken.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-71819
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 27 Oct 2022 10:36
• Thesis DOI: 10.5525/gla.thesis.71819
• URI: https://theses.gla.ac.uk/id/eprint/71819