Membrane proteins, including ion channels, make up nearly one-third of the proteome and constitute the majority of pharmaceutical drug targets. Yet their biophysical and biochemical characterization remains constrained by challenges in expression and stability. Advances in cell-free protein synthesis (CFPS) have enabled new workflows for the efficient production and stabilization of membrane proteins. By co-expressing target proteins with the scaffold protein ApoA1 and the lipid DMPC in scalable lysate systems, we achieve in vitro production of stable membrane protein complexes. Functional validation is achieved using electrophysiology platforms such as droplet interface bilayers (DIB), enabling characterization of voltage-gated ion channels such as CaV2.2 and NaV1.2. Specifically, DIB systems allowed us to determine functionality of these proteins as well as the change in their activity due to different buffer compostions and toxins. The integration of CFPS with bilayer-based electrophysiology establishes a versatile toolkit for biochemical and biophysical studies. Together, these methods provide a pathway to probe membrane protein dynamics, benchmark activity across membrane mimetics, and accelerate discovery in both basic science and therapeutic development.
Laurence et al. (Sun,) studied this question.