Expression of OmpG using in vitro translation system
OmpG, with or without addition of 20 mg/mL of a nano-sized liposome solution, was synthesized using a cell-free synthesis system. Fig. 1 shows the results of the SDS-PAGE analysis and western blot analysis of the OmpG synthesis solution. A band of OmpG was observed at approximately 30 kDa, which was not observed in the solution of the in vitro translation system without OmpG DNA (Figure 2 (a), Figure S1). The band of OmpG was detected by western blot analysis using an anti-His tag antibody because the His x 6 tag was conjugated at the N-terminal of OmpG (Figure 2 (b), Figure S2). Although the amount of OmpG synthesized in solution with the nano-sized liposomes was lower than that without the nano-sized liposomes, a sufficient amount of OmpG in the solution with nano-sized liposomes was also synthesized.
Incorporation of OmpG synthesized by the in vitro translation system into nano-sized liposomes
To investigate whether OmpG synthesized using the in vitro translation system was directly incorporated into the nano-sized liposome membrane, OmpG was synthesized into a cell-free synthesis solution containing various liposome compositions (DOPC, DLPC, DOPE/DOPG (7:3wt/wt%), or E. coli polar lipid extract (PE/PG/CA (67:23.2:9.8 wt/wt%))). After synthesis for 4 h at 37 °C, the synthesis of OmpG containing various liposome compositions was confirmed by SDS-PAGE. The bands of OmpG were detected in all samples containing nano-sized liposomes (Figure 3 (a), Figure S3 (a)). The bands of the samples were mixed in the OmpG-incorporated liposomes, the OmpG into the solution, and the components of cell-free synthesis. Therefore, to confirm the incorporation of OmpG into liposomes, liposomes containing OmpG were separated from the synthesized samples using a sucrose density gradient centrifugation method. The lipid concentrations in the purified liposome samples were determined by measuring the concentrations of PC and PE. The bands of OmpG from the purified liposome compositions were detected by SDS-PAGE analysis when the liposome solution at a constant lipid concentration was applied to the SDS-PAGE gel. The OmpG band was only detected at approximately 30 kDa. These band positions of the OmpG, including the nano-sized liposomes, corresponded to the position of the band of the OmpG with the native conformation, suggesting that the OmpG, which is incorporated into nano-sized liposomes, is the folded OmpG18 (Figure 3 (b), Figure S3 (b)). The amount of OmpG incorporated into the nano-sized liposomes was as follows: large amounts of OmpG were incorporated into E. coli lipid liposomes, followed in order by DOPE/DOPG (7:3) liposomes, DOPC liposomes, and DLPC liposomes. Compared to DOPC liposomes and DOPE/DOPG (7:3) liposomes, the amount of OmpG incorporated into the DOPE/DOPG (7:3) liposomes was larger than that into the DOPC liposomes (Figure 3 (b), Figure S3 (b)). The tendency of incorporation of OmpA into the nano-sized liposomes was the same as the amount of OmpG incorporated into the liposomes (Figure S4). Although the length of the fatty acid chain is the same, only DOPG has a negative charge. Compared with E. coli lipid liposomes and DOPE/DOPG (7:3) liposomes, the amount of OmpG incorporated into the E. coli lipid liposome was larger than that incorporated into the DOPE/DOPG liposomes. The surface charge of liposomes, including E. coli. lipid and DOPE/DOPG are negative, and the length of the fatty acid chain is different between the E. coli lipid (containing various lengths of the fatty acid chain) and DOPE/DOPG (containing one length of the fatty acid chain). These results indicate that the incorporation of OmpG influences the surface charge and variability in the length of the fatty acid chain of the liposomes. In previous studies of OmpA and OmpG synthesized by the in vitro translation system, the SDS-PAGE analysis of the OmpA and OmpG-containing liposomes that was not purified after the in vitro translation was difficult for estimating the amount of OmpA and OmpG incorporated into the nano-sized liposomes11,12. In this study, with the use of purified liposomes, the amount of OmpG incorporated in nano-sized liposomes, with various lipid components, was estimated for the first time. Although distinguishing between the OMPs embedded in the liposomes and those present free in the solution is difficult using previous methods, this experiment shows that the amount of OmpG inserted into the E. coli lipid liposomes was the largest, which were in the folded state without the presence of periplasmic chaperones such as Skp, DegP, and SurA.
Next, when the liposomes were added to the solution of the in vitro translation system before or after the synthesis of OmpG, the differences in the amount of OmpG incorporated into the E. coli lipid or DOPE/DOPG liposomes were investigated after the stepwise sucrose density gradient of the liposomes (Figure 3 (c), Figure S3 (b)). In case of the addition of the liposomes before the OmpG synthesis, the band of OmpG on the E.coli lipid or DOPE/DOPG liposomes was detected by SDS-PAGE analysis. In contrast, in the case of the addition of liposomes after OmpG synthesis, the band of OmpG on these liposomes was not detected. This result suggests that OmpG was incorporated into liposomes during OmpG synthesis. When membrane proteins were synthesized by the in vitro translation system, membrane proteins were incorporated into liposomes during their synthesis; in other words, the membrane proteins were aggregated in the absence of the liposomes during the cell-free synthesis14. Therefore, cell-free synthesis of functional membrane proteins requires the coexistence of liposomes. These results corresponded with previous results on membrane protein synthesis using an in vitro translation system19.
Electrophysiological measurement of OmpG on the nano-sized liposomes
To confirm the formation of nanopores of OmpG into the nano-sized liposomes, the current signals of the OmpG were measured by a BLM chip that was connected to a patch clamp amplifier20. The BLM was formed using the droplet contact method21. OmpG-containing liposomes were initially added to the droplets. By the fusion of liposomes with BLM, OmpG was incorporated into the BLM. We obtained an OmpG current amplitude of approximately 150 pA at +100 mV with 1 M KCl [Figures 4 (a) and (b)]. This amplitude was similar to that previously reported for OmpG channels in the BLM system22. This open-close signal shape is specific to OmpG. The specific current amplitude of OmpA in the nanosized liposomes was also detected (Figure S5). To the best of our knowledge, this is the first report of the ion current detection of Omp synthesized using an in vitro translation system. These results suggest that OmpG, which is incorporated into nano-sized liposomes, has a native conformation.