Advancing a genetically encoded cyclic peptide screening platform
Advancing a genetically encoded cyclic peptide screening platform
The effective combination of a bacterial Reverse Two-Hybrid System screening platform and a SICLOPPS randomised library has been previously used for the identification of cyclic peptide inhibitors of various protein-protein interactions.
A more robust Reverse Two-Hybrid System was designed and constructed to utilise a fluorescent protein reporter as well as an antibiotic resistance gene and HIS3 to enable selection of an inhibitor. The functionality of the new system was tested with the HIF1[alpha]/HIF1[Beta] and p6/UEV heterodimeric interactions and the cyclic peptide inhibitors that had been previously identified for these interactions.
Next, a split-intein with improved properties was used to construct a second generation SICLOPPS library, which was tested to show that it displayed more efficient splicing. The increased toxicity of the Nostoc punctiforme DnaE splitintein employed in the new library was reduced by the addition of the SsrA protein degradation tag to enable the identification of more varied cyclic peptide inhibitors in future SICLOPPS screens.
Townend, Jaime
598d5099-60ba-4a5b-89dc-b4c674c1aeda
30 September 2015
Townend, Jaime
598d5099-60ba-4a5b-89dc-b4c674c1aeda
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2
Townend, Jaime
(2015)
Advancing a genetically encoded cyclic peptide screening platform.
University of Southampton, Faculty of Natural and Environmental Sciences, Doctoral Thesis, 212pp.
Record type:
Thesis
(Doctoral)
Abstract
The effective combination of a bacterial Reverse Two-Hybrid System screening platform and a SICLOPPS randomised library has been previously used for the identification of cyclic peptide inhibitors of various protein-protein interactions.
A more robust Reverse Two-Hybrid System was designed and constructed to utilise a fluorescent protein reporter as well as an antibiotic resistance gene and HIS3 to enable selection of an inhibitor. The functionality of the new system was tested with the HIF1[alpha]/HIF1[Beta] and p6/UEV heterodimeric interactions and the cyclic peptide inhibitors that had been previously identified for these interactions.
Next, a split-intein with improved properties was used to construct a second generation SICLOPPS library, which was tested to show that it displayed more efficient splicing. The increased toxicity of the Nostoc punctiforme DnaE splitintein employed in the new library was reduced by the addition of the SsrA protein degradation tag to enable the identification of more varied cyclic peptide inhibitors in future SICLOPPS screens.
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Jaime Townend Thesis.pdf
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Published date: 30 September 2015
Organisations:
University of Southampton, Chemistry
Identifiers
Local EPrints ID: 398004
URI: http://eprints.soton.ac.uk/id/eprint/398004
PURE UUID: ea836bc5-4f63-4d30-a947-9292b59a37f3
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Date deposited: 15 Jul 2016 13:00
Last modified: 15 Mar 2024 05:44
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Author:
Jaime Townend
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