Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform

Cell-free transcription-translationplatforms havebeen widely utilized to express soluble proteins in basic synthetic biological circuit prototyping. From asynthetic biology point of view, it is critical to express membrane proteins in cell-freetranscription-translationsystems, and use them directly in biocircuits,considering the fact that histidine kinases, G-protein coupled receptors (GPCRs) and other important biosensors are all membraneproteins.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 solubilizedand activemembrane proteins in our cell-free TX-TL platform with membrane-like materials. Wefirsttested the expression ofseveral constructs with β1 and β2 adrenergicreceptorsin TX-TL and observed significantinsoluble membraneprotein production.The addition ofnanodiscs to the cell free expression system enabled solubilization of membrane proteins. Nanodisc is lipoprotein-based membrane-like material. The activity of β2 adrenergicreceptor was tested withboth fluorescence and Surface Plasmon Resonance (SPR) binding assays by monitoring the specific binding response ofsmall-molecule binders, carazolol and norepinephrine.Our results suggest that it is promisingto use cell-free expression systems to prototype synthetic biocircuits involvingsingle chain membrane proteinswithout extra procedures. This data made us one step closer to testingcomplex membrane protein circuits in cell-free environment.