Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells
Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host prot...
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my.iium.irep.966492022-03-03T00:23:41Z http://irep.iium.edu.my/96649/ Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells Isa, Nur Firdaus Bensaude, Olivier Murphy, Shona QR355 Virology RB Pathology Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss low-affinity and favour non-specific interactions. We have recently incorporated photoreactive amino acids at defined residues of a viral protein in vivo, by introducing amber stop codons (TAG) and using a suppressor tRNA. This is followed by UV-crosslinking, to identify interacting host proteins in live mammalian cells. The affinitypurified photo-crosslinked viral-host protein complexes are further characterized by mass spectrometry following extremely stringent washes. This combinatorial site-specific incorporation of a photoreactive amino acid and affinity purification-mass spectrometry strategy allows the definition of viral-host protein contacts at single residue resolution and greatly reduces non-specific interactors, to facilitate characterization of viral-host protein interactions Bio-protocol LLC 2022-02-05 Article PeerReviewed application/pdf en http://irep.iium.edu.my/96649/1/96649_Amber%20suppression%20technology%20for%20mapping%20site-specific.pdf application/pdf en http://irep.iium.edu.my/96649/7/96649_Amber%20suppression%20technology_Scopus.pdf Isa, Nur Firdaus and Bensaude, Olivier and Murphy, Shona (2022) Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells. Bio-protocol, 12 (3). E-ISSN 2331-8325 https://bio-protocol.org/e4315 10.3390/vaccines9101054 |
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QR355 Virology RB Pathology Isa, Nur Firdaus Bensaude, Olivier Murphy, Shona Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells |
description |
Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern
virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation
or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss
low-affinity and favour non-specific interactions. We have recently incorporated photoreactive amino acids at
defined residues of a viral protein in vivo, by introducing amber stop codons (TAG) and using a suppressor tRNA.
This is followed by UV-crosslinking, to identify interacting host proteins in live mammalian cells. The affinitypurified photo-crosslinked viral-host protein complexes are further characterized by mass spectrometry following
extremely stringent washes. This combinatorial site-specific incorporation of a photoreactive amino acid and affinity
purification-mass spectrometry strategy allows the definition of viral-host protein contacts at single residue
resolution and greatly reduces non-specific interactors, to facilitate characterization of viral-host protein interactions |
format |
Article |
author |
Isa, Nur Firdaus Bensaude, Olivier Murphy, Shona |
author_facet |
Isa, Nur Firdaus Bensaude, Olivier Murphy, Shona |
author_sort |
Isa, Nur Firdaus |
title |
Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells |
title_short |
Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells |
title_full |
Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells |
title_fullStr |
Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells |
title_full_unstemmed |
Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells |
title_sort |
amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells |
publisher |
Bio-protocol LLC |
publishDate |
2022 |
url |
http://irep.iium.edu.my/96649/1/96649_Amber%20suppression%20technology%20for%20mapping%20site-specific.pdf http://irep.iium.edu.my/96649/7/96649_Amber%20suppression%20technology_Scopus.pdf http://irep.iium.edu.my/96649/ https://bio-protocol.org/e4315 |
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