Almost all membrane proteins are inserted or translocated across membranes by the universally conserved Sec translocon. Despite its central role, experimental access to Sec function has remained limited. Here, we present ProSecCO (Protein Secretion in Cell-free via synthetic Operons), which is a cell-free protein synthesis platform that inserts SecYEG into synthetic vesicles, enabling direct testing of Sec in real-time and high-throughput, circumventing longstanding viability constraints. Screening 300 Sec variants in a single experiment, we consolidate three decades of Sec research, while vastly expanding mutant diversity for structure-function insights. Mapping over 30 functionally critical regions that modulate Sec activity across three orders of magnitude, we uncover dozens of super-active translocation variants and one variant of improved insertion activity. We further leverage ProSecCO to increase membrane protein quality and nanobody export, highlighting the potential of our system for advancing applications in synthetic biology and biotechnology.
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