Cell-free transcription-translation platforms have been widely utilized to express soluble proteins in basic synthetic biological circuit prototyping. From a synthetic biology point of view, it is critical to express membrane proteins in cell-free transcription-translation systems, and use them directly in biocircuits, considering the fact that histidine kinases, G-protein coupled receptors (GPCRs) and other important biosensors are all membrane proteins. Previous studies have expressed membrane proteins in cell-free systems with the help of detergents, liposomes or nanodiscs, but have not demonstrated the ability to prototype circuit behavior for the purpose of testing more complex circuit functions involving membrane-bound proteins. Built on previous efforts, in this work we demonstrated that we could co-translationally express solubilized and active membrane proteins in our cell-free TX-TL platform with membrane-like materials. We first tested the expression of several constructs with β1 and β2 adrenergic receptors in TX-TL and observed significant insoluble membrane protein production. The addition of nanodiscs to the cell free expression system enabled solubilization of membrane proteins. Nanodisc is lipoprotein- based membrane-like material. The activity of β2 adrenergic receptor was tested with both fluorescence and Surface Plasmon Resonance (SPR) binding assays by monitoring the specific binding response of small-molecule binders, carazolol and norepinephrine. Our results suggest that it is promising to use cell-free expression systems to prototype synthetic biocircuits involving single chain membrane proteins without extra procedures. This data made us one step closer to testing complex membrane protein circuits in cell-free environment.

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