Cell-free production of target-directed liposomes utilizing membrane localization peptides

Cell-free protein synthesis technology is a powerful tool for the rapid production of bioactive molecules such as antibodies. By combining with liposomes, immobilizing the synthesized proteins onto the liposome membrane leads production of bioactive liposomes within the cell-free reaction. These cell-free approaches can be applied in medical treatment and drug development, however, not many studies have been done to develop it as a highly versatile platform. Here, we explored hydrophobic amino acid sequences that lead hydrophilic proteins onto the liposome membrane and applied them to the co-translational production of bioactive liposomes. The membrane localizing peptides (MLPs) introduced at the N- or C-terminus of small molecular weight antibodies, nanobody, guided the membrane localization onto liposomes. The nanobodies-decorated liposomes specifically bound to cancer cells expressing the target antigen protein, thus suggesting maintaining functional structure on the liposome membrane. The MLPs we discovered can be widely applied to the rapid immobilization of proteins on liposome surfaces, leading to cell-free applications in virus-like particles or RNA therapy.