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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| Main Authors: | , , |
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| Format: | Article |
| Language: | en en |
| Published: |
Bio-protocol LLC
2022
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| Subjects: | |
| Online Access: | 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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| Summary: | 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 |
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