(629g) Invited Talk: Cell-Free Protein Synthesis Goes Electric: Measuring Transmembrane Ion Channel Activity Using Bioelectronic Circuits

Despite advancements in transmembrane protein (TMP) structural biology and a growing interest in their applications, this class of proteins remains challenging to study. Progress has been hindered by the complex nature of TMPs and innovative methods are required to circumvent technical hurdles. Cell-free protein synthesis (CFPS) is an emerging technique for synthesizing TMPs directly into a membrane environment using reconstituted components of the cellular transcription and translation machinery in vitro, bypassing purification and reconstitution. In this study, cell-free co-translational synthesis of an ion channel, MscL, into a supported lipid bilayer on a conducting polymer film is reported. Specifically, PEDOT:PSS films on ITO electrodes, which are both transparent and electrically conductive, allow the assessment of protein activity by optical and electrical means. Here, fluorescence microscopy and electrochemical impedance spectroscopy (EIS) are used to characterize the synthesized TMPs. Two different versions of MscL are used to detect a differential response to exposure of 2-(trimethylammonium)ethyl methanethiosulfonate, bromide (MTSET). The wild-type (WT-MscL) is not responsive to MTSET, while a cysteine modification enables the channel to open (G22C-MscL). This illustrates the ability to directly read out the activity of CFPS TMPs and discriminate them based on a differential electrical response. By dramatically simplifying TMP synthesis on a platform capable of sensing their biological function, we envision a future where large libraries of protein variants can be easily synthesized and assayed using bioelectronic circuits. Overlaying these advances with the massive parallelization possible with microfluidics, the ability to rapidly screen TMP activity will be scaled beyond what is currently feasible and open a broad spectrum of new applications.