Modular Cell-Free Extract Mixing Enables Soluble, Active Biomanufacturing of Difficult-to-Express and Aggregation-Prone Proteins

Cell-free protein synthesis (CFPS) is a powerful platform for rapid protein prototyping and biomanufacturing, but expression of complex proteins remains limited by labor-intensive extract preparation as well as the inability of conventional Escherichia coli extracts to efficiently support disulfide bond formation and prevent protein aggregation. We previously developed a Cell-Free Autoinduction (CFAI) workflow that simplifies extract preparation, however, CFAI-derived extracts retain the folding limitations of standard E. coli systems for producing traditionally difficult proteins. Here, we address these challenges by utilizing extracts enriched with molecular chaperones or Erv1p-based oxidative folding catalysts and combining them through a modular extract mixing strategy to enable independent tuning of the cell-free folding environment. This workflow supplies folding factors through enriched extracts, avoiding recurring purchases of purified supplements and enabling a modeled reagent-cost advantage. To evaluate real-world applications and utility of lysate mixing for protein production, we demonstrate a 10-fold increase in solubility of the aggregation-prone bacterial toxin Colicin M, achieving >95% soluble protein while retaining antimicrobial activity. We further used a Design of Experiments (DoE) framework to optimize combinations of chaperone-enriched and Erv1p-based oxidative folding extracts containing either protein disulfide isomerase (PDI) or DsbC. The optimized compositions produced >95% soluble truncated human tissue plasminogen activator (vtPA), comprising the kringle 2 and protease domains, while preserving enzymatic activity. Together, these results demonstrate that CFAI-derived modular extract mixing provides a tunable, high-throughput strategy with low modeled materials costs for tailoring cell-free biochemical environments for difficult-to-express proteins, expanding CFPS utility for protein engineering and biomanufacturing.