Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells

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

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Abstract

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

Item Type:	Article (Journal)
Uncontrolled Keywords:	Suppressor tRNA, Aminoacyl tRNA synthetase, P-benzoylphenylalanine (Bpa), Photoactivated protein-protein crosslinking, Unnatural amino acid, Amber stop codon
Subjects:	Q Science > QR Microbiology > QR355 Virology R Medicine > RB Pathology
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button):	Kulliyyah of Science > Department of Biotechnology Kulliyyah of Science
Depositing User:	Dr Nur Firdaus Isa
Date Deposited:	10 Feb 2022 14:54
Last Modified:	03 Mar 2022 08:23
URI:	http://irep.iium.edu.my/id/eprint/96649

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