(429b) Characterizing and Enriching Native Membrane Vesicles in E. coli Extracts to Improve Cell-Free Glycoprotein Synthesis

Cell-free protein synthesis harnesses endogenous transcription and translation machinery in cell extracts to enable biomanufacturing without living cells. To date, E. coli cell-free systems have been used for many applications including decentralized manufacturing of therapeutics and vaccines. Notably, key biopharmaceutical products such as conjugate vaccines rely on protein glycosylation, a post-translational modification that requires membrane-bound machinery. To tailor E. coli cell-free reactions to make specific products, soluble heterologous proteins are routinely expressed in the strain prior to lysis. However, membrane-bound functionality has been largely inaccessible in extracts without synthetic membrane mimics. In this work we characterize native membrane vesicles in cell extracts for applications in membrane-dependent protein glycosylation. We first characterize the size and concentration of native vesicles that form during cell lysis. We then determine how extract processing steps impact vesicle properties and create simple methods for modulating concentrations of membrane vesicles and their heterologous cargo. By tuning upstream processing conditions, we increase vesicle concentration in extract by up to 2-fold and use vesicles to enrich a variety of membrane proteins in extract. We show that heterologous membrane-bound glycosylation machinery from the periplasm is associated with membrane vesicles and by applying extract processing improvements, we increase glycoprotein yields by up to 90%. Overall, this work provides valuable characterization of E. coli extracts and expands opportunities for engineering membrane-bound functionality in cell-free systems.