In situ Membrane Protein Expression by Efficient Recruitment of mRNA to the Membranes of Synthetic Cells

The synthesis of artificial cells is crucial for understanding the origins of life. In synthetic cells, however, the absence of membrane-bound organelles and auxiliary proteins severely limits the efficient expression and precise localization of membrane proteins using cell-free expression systems. Here we introduce a robust method that significantly enhances membrane protein synthesis by recruiting mRNA to phospholipid membranes. This is achieved through the use of cholesterol-modified, single-stranded DNA that anchors to the membrane and pairs with the mRNA’s untranslated region. This strategic placement facilitates the assembly of protein expression machinery, promoting direct co-translational folding at the membrane. Our approach not only ensures correct protein topology and functionality but also demonstrates broad applicability for the in situ expression of various membrane proteins. It effectively addresses the challenges of membrane protein localization and assembly in synthetic cells, showcasing its versatility for synthesizing a wide array of membrane proteins.