In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes.

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach.

Integrating reductive and synthetic approaches in biology using man-made cell-like compartments.

We propose 'integrated synthetic genetics' as a novel methodology that integrates reductive and synthetic approaches used in life science research. Integrated synthetic genetics enables determinations of sets of genes required for the functioning of any biological subsystem. This method utilizes artificial cell-like compartments, including a randomly introduced whole gene library, strictly defined components for in vitro transcription and translation and a reporter that fluoresces 'only when a particular function of a target biological subsystem is active.' The set of genes necessary for the target biological subsystem can be identified by isolating fluorescent artificial cells and multiplex next-generation sequencing of genes included in these cells. The importance of this methodology is that screening for the set of genes involved in a subsystem and reconstructing the entire subsystem can be done simultaneously. This methodology can be applied to any biological subsystem of any species and may remarkably accelerate life science research.