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. 2023:186:103-120.
doi: 10.1007/10_2023_228.

Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes

Yogesh Pandey  1   2 , Srujan Kumar Dondapati  3 , Doreen Wüstenhagen  1 , Stefan Kubick  1   4   5   6
Affiliations

Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes

Yogesh Pandey et al. Adv Biochem Eng Biotechnol. 2023.

Abstract

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.

Keywords: Cell-free protein synthesis; Eukaryotic lysates; Ion channels; Membrane proteins; Microsomes; Planar lipid bilayer.

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