Synthesizing and Expressing Native Ion Channels.

Abstract

Synthesizing and expressing ion channels in heterologous systems enable the characterization of the functional properties of these proteins. The cDNA that encodes ion channels can be amplified directly from mRNA or synthesized de novo in its entirety before cloning into an appropriate expression vector. Gibson assembly is a powerful tool that allows rapid cloning and integration of protein-coding cDNA into a variety of expression vectors. Here we describe a method in which the cDNA encoding a native snake ion channel (NaV 1.4) is synthesized in four equal-sized pieces (or blocks), and then assembled and ligated into an expression vector. Once in an appropriate expression vector, the assembled cDNA can be used for synthesis of mRNA, and the mRNA injected and expressed in Xenopus oocytes. This method has significant advantages over traditional rtPCR and ligation-based cloning including speed, cost, ease of codon optimization, and inclusion of silent restriction sites for Gibson-based mutagenesis.