In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system

Abstract

This chapter has adapted the nonsense codon suppression method for the incorporation of unnatural amino acids into membrane proteins in a Xenopus oocyte expression system. Combining this method with electrophysiologic analysis allows it to probe structure-function relationships in ion channels and receptors in ways not possible with conventional mutagenesis. In the absence of atomic-scale, structural data for membrane proteins, these techniques can provide detailed structural information. In the nonsense codon suppression method, Xenopus oocytes are coinjected with two RNAs: (1) mRNA transcribed in vitro from a mutated cDNA containing a TAG nonsense (stop) codon at the position of interest and (2) a suppressor tRNA containing the corresponding anticodon, CUA, and chemically acylated at the end with an amino acid. During protein synthesis, the aminoacylated suppressor tRNA directs the incorporation of the amino acid into the desired position of the protein. Because the amino acid is appended synthetically, it is not limited to the natural, and many unnatural amino acids have been incorporated into various proteins using this method. This chapter describes suppressor tRNA design and synthesis, chemical acylation of the suppressor tRNA, relevant organic synthesis methods, and optimization of mRNA and suppressor tRNA for Xenopus oocyte expression.