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Development of a novel two-hybrid system for the identification of cyclic peptide induced protein-protein association

Development of a novel two-hybrid system for the identification of cyclic peptide induced protein-protein association
Development of a novel two-hybrid system for the identification of cyclic peptide induced protein-protein association
Chemically-induced protein association is involved in key regulatory pathways throughout the body. Many natural products exert their downstream effect by the stabilisation of naturally occurring interactions, either by an allosteric effect or by the direct binding of two distinct protein partners. The targeting of these interactions by small-molecules is a relatively under developed field, with the inhibition of existing protein-protein interactions (PPIs) a more investigated target. One such method to investigate PPI inhibition is a bacterial reverse two-hybrid system (RTHS), which has been successfully coupled to a split-intein based library of cyclic peptides to identify novel inhibitors (split-intein circular ligation of peptides and proteins, SICLOPPS). Here, the existing RTHS technology was rebuilt to enable the screening of cyclic peptides for their ability to induce dimerisation of two target proteins.

The well-studied mTOR-FKBP12 interaction, with heterodimerisation induced by the natural product rapamycin, was used to build and validate a novel two-hybrid system. This utilises the tetracycline repressor to reverse the transcriptional control from the RTHS, leading to a selectable phenotype upon dimerisation. Rapamycin addition successfully results in the association of these target proteins, leading to the downstream expression of two essential reporter genes and a fluorescent protein. This rapamycin-dependent interaction can be identified both by an agarbased growth assay, and a liquid media-based fluorescent assay.

A large number of SICLOPPS libraries were screened with little success at replicating the action of brapamycin. The SICLOPPS technology was optimised and expanded, enabling an improved screening platform for the future.
University of Southampton
Foster, Andrew
28b09ecd-0453-4dd5-bd5e-77504827bd03
Foster, Andrew
28b09ecd-0453-4dd5-bd5e-77504827bd03
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2

Foster, Andrew (2017) Development of a novel two-hybrid system for the identification of cyclic peptide induced protein-protein association. University of Southampton, Doctoral Thesis, 222pp.

Record type: Thesis (Doctoral)

Abstract

Chemically-induced protein association is involved in key regulatory pathways throughout the body. Many natural products exert their downstream effect by the stabilisation of naturally occurring interactions, either by an allosteric effect or by the direct binding of two distinct protein partners. The targeting of these interactions by small-molecules is a relatively under developed field, with the inhibition of existing protein-protein interactions (PPIs) a more investigated target. One such method to investigate PPI inhibition is a bacterial reverse two-hybrid system (RTHS), which has been successfully coupled to a split-intein based library of cyclic peptides to identify novel inhibitors (split-intein circular ligation of peptides and proteins, SICLOPPS). Here, the existing RTHS technology was rebuilt to enable the screening of cyclic peptides for their ability to induce dimerisation of two target proteins.

The well-studied mTOR-FKBP12 interaction, with heterodimerisation induced by the natural product rapamycin, was used to build and validate a novel two-hybrid system. This utilises the tetracycline repressor to reverse the transcriptional control from the RTHS, leading to a selectable phenotype upon dimerisation. Rapamycin addition successfully results in the association of these target proteins, leading to the downstream expression of two essential reporter genes and a fluorescent protein. This rapamycin-dependent interaction can be identified both by an agarbased growth assay, and a liquid media-based fluorescent assay.

A large number of SICLOPPS libraries were screened with little success at replicating the action of brapamycin. The SICLOPPS technology was optimised and expanded, enabling an improved screening platform for the future.

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Foster final thesis - Version of Record
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Published date: November 2017

Identifiers

Local EPrints ID: 418010
URI: http://eprints.soton.ac.uk/id/eprint/418010
PURE UUID: e6ad9aaf-caaf-4071-ba91-cd5997a170b9
ORCID for Ali Tavassoli: ORCID iD orcid.org/0000-0002-7420-5063

Catalogue record

Date deposited: 20 Feb 2018 17:30
Last modified: 29 Jan 2021 05:01

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Contributors

Author: Andrew Foster
Thesis advisor: Ali Tavassoli ORCID iD

University divisions

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