Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte

• Published in: Scientific reports Vol. 8; no. 1; pp. 5166 - 6
• Main Authors: Infield, Daniel T., Lueck, John D., Galpin, Jason D., Galles, Grace D., Ahern, Christopher A.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 26-03-2018; Nature Publishing Group
• Subjects: 631/337; 631/92/269; 631/92/269/1152; Acylation; Amino acids; Aminoacylation; Genetic code; Humanities and Social Sciences; Lysine; Membrane proteins; multidisciplinary; Protein structure; Science; Science (multidisciplinary); Sodium; Sodium channels (voltage-gated); tRNA; Xenopus
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-23201-z

Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the
Xenopus laevis
oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA
(pylT)
from Methanosarcina
barkeri fusaro
is orthogonal and highly competent for genetic code expansion experiments in the
Xenopus
oocyte. The data show that
pylT
is amendable to chemical acylation
in vitro
; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the
pylT
is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus,
pylT
is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the
Xenopus
oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.