Transporter function characterization via continuous-exchange cell-free synthesis and solid supported membrane-based...

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H⁺-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na⁺/H⁺ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as K_M, I_MAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.