Voltage-gated ion channels are a class of membrane proteins that temporally orchestrate the ion flux critical for chemical and electrical signaling in excitable cells. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes.
Current methods to investigate the function of these channels rely on heterologous expression in living systems or reconstitution into artificial membranes; however these approaches have inherent drawbacks which limit potential biophysical applications. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes. In this method, proteoliposomes containing Shaker potassium channels are synthesized in vitro and injected into the oocytes, yielding functional preparations as shown by electrophysiological and fluorescence measurements within few hours. This strategy for studying eukaryotic ion channels is contrasted with existing, laborious procedures that require membrane protein extraction and reconstitution into synthetic lipid systems.
Fig. 1 from Jarecki, et al., 2013: Cartoon workflow diagram of cell-free protein production and expression in Xenopus laevis oocytes illustrated in two phases. Phase 1: Unique 5' and 3' restriction sites appended onto a target gene open-reading frame are used to digest and subclone into a specialized plasmid (pEU-HSBC) for cell-free synthesis. The plasmid DNA is used as a template for in vitro transcription of RNA. Transcribed mRNA directs protein translation in wheat-germ cell-free extract supplemented with liposomes to make proteoliposomes. Since the Shaker channels can be recovered from the pellet fraction, the supernatant is discarded and the proteoliposomes are collected after centrifugation. Phase 2: Samples are reconstituted to specified concentrations in a salt buffer and injecting into single Xenopus laevis oocytes at the vegetal equator. Currents are measured under voltage-clamp within 24 hrs.
Jarecki BW, Makino S, Beebe ET, Fox BG, Chanda B. "Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes." Sci. Rep. 3:1040 (2013). PubMed ID: 23301161 | Search SBKB Publications portal | PMC Link
Brian G. Fox (firstname.lastname@example.org)
Transmembrane Protein Center
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Last edited:Mon 10 Mar 2014 - 5 years, 1 month ago