Expanding the Genetic Code of Xenopus laevis Embryos

Abstract

The incorporation of unnatural amino acids into proteins through genetic code expansion has been successfully adapted to African claw-toed frog embryos. Six unique unnatural amino acids are incorporated site-specifically into proteins and demonstrate robust and reliable protein expression. Of these amino acids, several are caged analogues that can be used to establish conditional control over enzymatic activity. Using light or small molecule triggers, we exhibit activation and tunability of protein functions in live embryos. This approach was then applied to optical control over the activity of a RASopathy mutant of NRAS, taking advantage of generating explant cultures from Xenopus. Taken together, genetic code expansion is a robust approach in the Xenopus model to incorporate novel chemical functionalities into proteins of interest to study their function and role in a complex biological setting.

Genetically Encoded Lysine Analogues with Differential Light Sensitivity for Activation of Protein Function

“…[34] We used the MbPylRS/PylT pair from Methanosarcina barkeri, an archaeal species; this system is orthogonal in both prokaryotes and eukaryotes including bacterial, yeast, and mammalian cells, [10,43,44] as well as fruit flies, nematodes, frogs, mice, and zebrafish. [45][46][47][48][49] This pair functions orthogonally to native tRNAs and aminoacyl-tRNA synthetases. The anticodon of PylT, CUA, enables genetic code expansion by suppressing in-frame amber stop codons (UAG).…”