Cell-free synthesis of cytochrome bo₃ ubiquinol oxidase in artificial membranes

The analysis of membrane proteins is notoriously difficult because isolation and detergent-mediated reconstitution often results in compromising the protein structure and function. We introduce a novel strategy of combining a cell-free expression method for synthesis of a protein species coping with one of the most important obstacles in membrane protein research - preserving the structural-functional integrity of a membrane protein species and providing a stable matrix for application of analytical tools to characterize the membrane protein of interest. We address integration and subsequent characterization of the cytochrome bo₃ ubiquinol oxidase (Cyt-bo₃) from de novo synthesis without the effort of conventional cell culture, isolation, and purification procedures. The experimental output supports our idea of a suitable platform for in vitro protein synthesis and functional integration into a membrane-mimicking structure. We show the compatibility of different concepts of in vitro synthesis toward biosensor applicability by the example of Cyt-bo ₃ protein expression. Our results obtained from in vitro synthesized proteins displayed similar behavior to proteins isolated from the cellular context. Overall, our approach is suitable for the in vitro expression of "complex" protein species such as Cyt-bo₃, which can be reproducible and stably synthesized and preserved in robust, synthetic planar membrane architecture.