Abstract
Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function (Brown et al., 2018; Chen et al., 2017; Chin et al., 2003; Wang, 2017). Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with UAG and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA (Ibba and Hennecke, 1995; Kavran et al., 2007). Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs Diazirine-lysine (AbK), Azido-lysine (AzK), and bicyclononyne-lysine (BCNK), 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 the UNAA mutagenesis to investigate and engineer protein function in zebrafish.