Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Abstract

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 this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Figure 1:. Experimental steps of inverted emulsion.

(1) Step 2.3.1 through step 2.3.3 of the protocol are visualized to demonstrate the assembly of the lipid monolayer at the interface of the lipid-oil mix and outer buffer solution. (2) Visualization of step 2.3.5 of the protocol is shown here to represent the formation of the lipid monolayer around emulsified droplets encapsulating the inner CFE solution. (3) Step 2.3.6 of the protocol shows the addition of the monolayer GUVs to the microcentrifuge tube with the lipid monolayer at the interface of a lipid-oil mix and outer buffer solution. (4) Step 2.3.7 is depicted here, in which centrifugation leads to the formation of a GUV pellet in the outer solution. (5) Step 2.3.8 is shown here, indicating the process of removing the excess lipid-in-oil mixture and outer solution. (6) Finally, step 2.3.9 is depicted here, where the GUV pellet is resuspended in the outer solution, and the GUVs are ready for incubation, followed by imaging.

Figure 2:. Protein expression in bulk CFE reactions and in GUVs encapsulating CFE reactions.

(A) Fluorescence readouts of individual bulk CFE reactions expressing WT-GluR0-sfGFP, PRSP-GluR0-sfGFP, and soluble sfGFP. The soluble sfGFP graph represents the signal from a 2.5 μL reaction (standard reaction volume is 20 μL) to avoid oversaturation of the plate reader measurements. Data is presented as mean ± S.D, n = 3. (B) Left: A representative confocal image of a GUV encapsulating CFE reaction expressing WT-GluR0-sfGFP. Middle: A representative confocal image of GUVs encapsulating CFE reaction expressing PRSP-GluR0-sfGFP. Right: A representative confocal image of a GUV encapsulating CFE reaction expressing soluble sfGFP. Scale bars: 10 μm.