Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements.

To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle-vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5-20 µm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30 min, 1 h, 2 h, and 4 h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3'-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches.

Control of Enzyme Reaction Initiation inside Giant Unilamellar Vesicles by the Cell-Penetrating Peptide-Mediated Translocation of Cargo Proteins.

Cell-penetrating peptides (CPPs) play important roles in directly delivering biomolecules, such as DNA, proteins, and peptides, into living cells. In artificial lipid membranes, such as planar lipid bilayers, the direct membrane translocation of ß-galactosidase via Pep-1 (one of the CPPs) is dependent upon a voltage gradient between the inner and outer leaflets of the lipid membranes. Giant unilamellar vesicles (GUVs) with asymmetric lipid distributions, which are recently generated using microfluidic technologies, can be observed by optical microscopy. Therefore, interactions between CPPs and asymmetric lipid bilayers in different kinds of lipids and the translocation mechanism of proteins via CPPs into GUVs can be investigated at the level of a single asymmetric GUV. This CPP-based system for transporting proteins into GUVs will be applied to control the start of enzyme reactions in GUVs. This study aimed to explore efficient protein translocation into GUVs via CPP and demonstrate that enzymatic reactions start in GUVs using a CPP-mediated direct translocation. The interactions and the enzyme reactions between the CPP (Pep-1 or penetratin)-DNase I complexes and the asymmetric or symmetric GUV membranes containing the negatively or neutrally charged lipids were observed by confocal laser-scanning microscopy. The asymmetric GUVs containing phosphatidylserine (PS) in the inner leaflet showed efficient DNase I translocation into GUVs via penetratin. Finally, the formation of a cross-linked actin network was observed in asymmetric PS GUVs incubated with Pep-1-streptavidin complexes. The CPP-mediated direct translocation can contribute to developing artificial cell models with the capacity to control the initiation of enzymatic reactions.