Cell-Free Protein Synthesis inside Giant Unilamellar Vesicles Analyzed by Flow Cytometry

Abstract

Lipid vesicles have been used as model cell systems, in which an in-vitro transcription-translation system (IVTT) is encapsulated to carry out intravesicular protein synthesis. Despite a large number of previous studies, a quantitative understanding of how protein synthesis inside the vesicles is affected by the lipid membrane remains elusive. This is mainly because of the heterogeneity in structural properties of the lipid vesicles used in the experiments. We investigated the effects of the phospholipid membrane on green fluorescent protein (GFP) synthesis occurring inside cell-sized giant unilamellar vesicles (GUV), which have a defined quantity of lipids relative to the reaction volume. We first developed a method to distinguish GUV from multilamellar vesicles using flow cytometry (FCM). Using this method, we investigated the time course of GFP synthesis using one of the IVTT, the PURE system, and found that phospholipid in the form of GUV has little effect on GFP synthesis based on three lines of investigation. (1) GFP synthesis inside the GUV was not dependent on the size of GUV (2) or on the fraction of cholesterol or anionic phospholipid constituting the GUV, and (3) GFP synthesis proceeded similarly in GUV and in the test tube. The present results suggest that GUV provides an ideal reaction environment that does not affect the internal biochemical reaction. On the other hand, we also found that internal GFP synthesis is strongly dependent on the chemical composition of the outer solution.

In vitro evolution of α-hemolysin using a liposome display

“…The difference in the vesicle volume may affect the intravesicular protein synthesis, for example, by creating a difference in the ratio of the surface area to the volume. This was not the case, because our previous report indicated that GFP synthesis using 1 nM DNA inside the GUV proceeded similarly regardless of the vesicle volume (21). Furthermore, the volume distribution of the vesicle did not change significantly during the protein synthesis (5-h incubation at 37°C) (Fig.…”

“…ii) Conditions appropriate for the target membrane protein can be adopted because the lipid composition of the GUVs is adjustable. We have shown previously that GUVs with different lipid compositions can be used without affecting intravesicular protein synthesis (21), indicating that a wide range of membranes may be used for liposome display. iii) Conditions appropriate for the target membrane protein can be adopted because the intravesicular solution can be adjusted as needed.…”

“…GUVs containing the PURE system were prepared using the water-in-oil (w/o) emulsion/transfer method (16,43), as described in our previous report (21). Briefly, 30 μL of the PURE system supplemented with the template DNA, 200 mM sucrose, 0.8 U/μL RNase inhibitor (RNasIn Plus; Promega), 1 μM transferrin Alexa Fluor 647 conjugate (TA647; Life Technologies), and 3 μM HaloTag protein (SI Materials and Methods) was added to 300 μL of liquid paraffin (Wako Pure Chemical Industries) containing 3 mg of POPC (Avanti Polar Lipids) and 3 mg of cholesterol (Nacalai Tesque).…”

“…The conversion was performed using the equation V = 0.0038 FI 647 , where V (femtoliters) is the volume of the GUVs and FI 647 is the TA647 fluorescence intensity. Analysis and sorting were conducted with vesicles that satisfied log(FSC-A) > 1.5 × log(SSC-A) − 1, which is the population defined as the GUVs in our previous reports (16,21).…”

On Fine Stochastic Simulations of Liposome-Encapsulated PUREsystem™

“…Other numerous applications of the compartmentalized PS were focused on understanding gene expression inside liposomes, such as in the presence of different membrane lipid composition [10], with different preparation methods [11], and in different-sized vesicles [12].…”