Abstract Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli -based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3×10 12 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7×10 12 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N -linked and O -linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles