biostudies-literatureUnknownLoi KJNIBIB NIH HHSNIAMS NIH HHSCell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing Journal of visualized experiments : JoVEhttps://www.ebi.ac.uk/biostudies/studies/S-EPMC12289319biostudies-literatureReconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.PMC12289319R01 EB030031R21 AR080363Loi KJLiu APMoghimianavval HfalseReconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing 2024-01-01T00:00:00Z2024 Mar2026-03-15T18:18:27.696Z2025-08-13T03:04:30.089ZS-EPMC122893193852608710.3791/66595