Expression of Human CYP2D6 in Artificial Cells Based on Polymer‐Lipid Hybrid Vesicles

Artificial cells with the ability to express functional, biomedically relevant enzymes have long been a goal in the field of bottom-up synthetic biology. Here, we demonstrate the expression of human cytochrome P450 2D6 (CYP2D6) within the lumen of giant hybrid vesicle-based artificial cells using a cell-free protein expression system based on the protozoan Leishmania tarentolae. The vesicle membranes comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine and amphiphilic block copolymers containing poly(cholesteryl methacrylate-co-butyl methacrylate) with either poly(galactose methacrylate) (pGalacMA) or poly(carboxyethyl acrylate) as the hydrophilic block. The amount and composition of the block copolymer incorporated into the giant hybrid vesicle membrane altered the artificial cells' ability to support functional protein expression, likely through changes in membrane properties. In particular, giant hybrid vesicles containing pGalacMA supported production of enzymatically active CYP2D6. Overall, the reported approach advances the development of artificial cells capable of producing functional, biomedically relevant enzymes. Further, the effort highlights complex interplay between polymer properties and performance of biological components in the functional complexity achievable in polymer-lipid hybrid vesicles.