Development of a fluorescent three‐hybrid system for the identification of protein‐protein associators
Development of a fluorescent three‐hybrid system for the identification of protein‐protein associators
The chemically‐induced dimerization of proteins is fundamental to many key regulatory pathways. A number of natural products exert their downstream effect through the stabilization of a protein complex, either by direct binding to two distinct protein partners or via an allosteric mechanism. Here, we report a bacterial three‐hybrid system, with dual life/death and fluorescent reporters, which detects protein association and is compatible with high‐throughput screening. We use rapamycin mediated mTOR‐FKBP12 (mammalian target of rapamycin—FK506 Binding Protein 12 kDa) dimerization to validate this platform; the addition of rapamycin results in the association of these target proteins, leading to the expression of two essential life/death reporter genes and a fluorescent signal. We further used this system to quantify the activity of rapamycin by utilizing the fluorescent readout, exemplifying its potential in screening and for ranking large in vivo libraries for compounds that upregulate the association of any two given proteins.
protein-protein interaction, rapamycin, two-hybrid system
Foster, Andrew D.
e12c139f-cb45-4bed-ae80-821e9b540f3d
Chung, Chun‐wa
82aac1f7-0075-4618-8bca-e21f5eb65e81
Hann, Michael M.
28a9fd0c-08ae-4241-9ee6-3a6684e04768
Simpson, Graham L.
f714627d-b9f3-4b2f-b4c3-516474e8a33f
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2
5 January 2021
Foster, Andrew D.
e12c139f-cb45-4bed-ae80-821e9b540f3d
Chung, Chun‐wa
82aac1f7-0075-4618-8bca-e21f5eb65e81
Hann, Michael M.
28a9fd0c-08ae-4241-9ee6-3a6684e04768
Simpson, Graham L.
f714627d-b9f3-4b2f-b4c3-516474e8a33f
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2
Foster, Andrew D., Chung, Chun‐wa, Hann, Michael M., Simpson, Graham L. and Tavassoli, Ali
(2021)
Development of a fluorescent three‐hybrid system for the identification of protein‐protein associators.
Peptide Science, 113 (1), [e24178].
(doi:10.1002/pep2.24178).
Abstract
The chemically‐induced dimerization of proteins is fundamental to many key regulatory pathways. A number of natural products exert their downstream effect through the stabilization of a protein complex, either by direct binding to two distinct protein partners or via an allosteric mechanism. Here, we report a bacterial three‐hybrid system, with dual life/death and fluorescent reporters, which detects protein association and is compatible with high‐throughput screening. We use rapamycin mediated mTOR‐FKBP12 (mammalian target of rapamycin—FK506 Binding Protein 12 kDa) dimerization to validate this platform; the addition of rapamycin results in the association of these target proteins, leading to the expression of two essential life/death reporter genes and a fluorescent signal. We further used this system to quantify the activity of rapamycin by utilizing the fluorescent readout, exemplifying its potential in screening and for ranking large in vivo libraries for compounds that upregulate the association of any two given proteins.
Text
pep2.24178
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More information
Accepted/In Press date: 22 May 2020
e-pub ahead of print date: 5 June 2020
Published date: 5 January 2021
Additional Information:
Funding Information:
This work was funded by the Engineering and Physical Sciences Research Council and GlaxoSmithKline (iCASE studentship for A.F.).
Publisher Copyright:
© 2020 The Authors. Peptide Science published by Wiley Periodicals LLC
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
Keywords:
protein-protein interaction, rapamycin, two-hybrid system
Identifiers
Local EPrints ID: 441512
URI: http://eprints.soton.ac.uk/id/eprint/441512
ISSN: 2475-8817
PURE UUID: 72df832a-d47a-451d-bc12-b9ec2a155e51
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Date deposited: 16 Jun 2020 16:34
Last modified: 17 Mar 2024 03:07
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Contributors
Author:
Andrew D. Foster
Author:
Chun‐wa Chung
Author:
Michael M. Hann
Author:
Graham L. Simpson
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