Solubilization of Oligomeric Cell-Free Synthesized Proteins Using SMA Copolymers.

Although membrane proteins are abundant in nature, their investigation is limited due to bottlenecks in heterologous overexpression and consequently restricted accessibility for downstream applications. In this chapter, we address these challenges by presenting a fast and straightforward synthesis platform based on eukaryotic cell-free protein synthesis (CFPS) and an efficient solubilization strategy using styrene-maleic acid (SMA) copolymers. We demonstrate CFPS of TWIK-1, a dimeric ion channel, based on Sf21 (Spodoptera frugiperda) insect lysate showing homooligomerization and N-glycosylation enabled by endoplasmic reticulum-derived microsomes. Furthermore, we employ SMA copolymers for protein solubilization, which preserves the native-like microsomal environment. This approach not only retains the solubilized protein's suitability for downstream applications but also maintains the oligomerization and glycosylation of TWIK-1 post-solubilization. We validate the solubilization procedure using autoradiography, particle size analysis, and biomolecular fluorescence assay and confirm the very efficient, structurally intact solubilization of cell-free synthesized TWIK-1.

Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K2P.

Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, in this work, we demonstrate the cell-free synthesis (CFS) as an alternative platform to study the assembly of two-pore domain potassium channels (K2P) within endogenous endoplasmic reticulum-derived microsomes. Exploiting the open nature of CFS, we investigate the cotranslational translocation of TREK-2 into the microsomes and suggest a cotranslational assembly with typical single-channel behavior in planar lipid-bilayer electrophysiology. The heteromeric assembly of K2P channels is a contentious matter, accordingly we prove the successful assembly of TREK-2 with TWIK-1 using a biomolecular fluorescence complementation assay, Western blot analysis and autoradiography. The results demonstrate that TREK-2 homodimer assembly is the initial step, followed by heterodimer formation with the nascent TWIK-1, providing evidence of the intergroup heterodimerization of TREK-2 and TWIK-1 in eukaryotic CFS. Since K2P channels are involved in various pathophysiological conditions, including pain and nociception, CFS paves the way for in-depth functional studies and related pharmacological interventions. This study highlights the versatility of the eukaryotic CFS platform for investigating ion channel assembly in a native-like environment.