Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

• Main Authors: Junetha Syed, Saravanan Palani, Scott T. Clarke, Zainab Asad, Andrew R. Bottrill, Alexandra M.E. Jones, Karuna Sampath, Mohan K. Balasubramanian
• Format: Article
• Language: English
• Published: MDPI AG 2019-05-01
• Series: International Journal of Molecular Sciences
• Subjects: genetic code expansion; zebrafish; unnatural amino acid; protein engineering; proteomics; crosslinking; click chemistry
• Online Access: https://www.mdpi.com/1422-0067/20/10/2577

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-<span style="font-variant: small-caps;">s</span>-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.