Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 20, Issue 10, Pages -Publisher
MDPI
DOI: 10.3390/ijms20102577
Keywords
genetic code expansion; zebrafish; unnatural amino acid; protein engineering; proteomics; crosslinking; click chemistry
Funding
- Wellcome Trust Senior Investigator Award [WT101885MA]
- Royal Society Wolfson merit award [WM130042]
- European Research Council Advanced Grant (ERC-2014-ADG) [671083]
- Biotechnology and Biological Sciences Research Council
- Engineering and Physical Sciences Research Council [EP/F500378/1]
- European Research Council (ERC) [671083] Funding Source: European Research Council (ERC)
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Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNA(CUA) that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNA(CUA) with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.
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