Modular Cell-Free Extract Mixing Enables Soluble, Active Biomanufacturing of Difficult-to-Express and Aggregation-Prone Proteins
- 1California Polytechnic State University
Abstract
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.
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BibTeX
@misc{firmansyah2026modular,
title = {{Modular Cell-Free Extract Mixing Enables Soluble, Active Biomanufacturing of Difficult-to-Express and Aggregation-Prone Proteins}},
author = {L. Firmansyah and A. Delmundo and J. P. Oza},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.09.757855},
url = {https://www.biorxiv.org/content/10.64898/2026.10.09.757855v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Modular Cell-Free Extract Mixing Enables Soluble, Active Biomanufacturing of Difficult-to-Express and Aggregation-Prone Proteins
AU - L. Firmansyah
AU - A. Delmundo
AU - J. P. Oza
PY - 2026
DA - 2026/10/09
PB - bioRxiv
DO - 10.64898/2026.10.09.757855
UR - https://www.biorxiv.org/content/10.64898/2026.10.09.757855v1
AB - 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.
ER -