{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "California Polytechnic State University"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 3,
  "authors": [
    {
      "name": "L. Firmansyah"
    },
    {
      "name": "A. Delmundo"
    },
    {
      "affs": [
        0
      ],
      "name": "J. P. Oza"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/9rpsqxgq0s3f6.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.09.757855",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.09.757855v1.full",
  "id": "9rpsqxgq0s3f6",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.09.757855v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "synthetic biology"
    ],
    [
      "openalex",
      "1312|RNA and protein synthesis mechanisms"
    ]
  ],
  "openalex": "W7221506416",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.09.757855v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-09",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/9rpsqxgq0s3f6.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:27:19Z",
      "native_id": "10.64898/2026.10.09.757855",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.09.757855v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:synthetic biology",
      "subfield": "systems-synthetic"
    }
  ],
  "title": "Modular Cell-Free Extract Mixing Enables Soluble, Active Biomanufacturing of Difficult-to-Express and Aggregation-Prone Proteins",
  "title_text": "Modular Cell-Free Extract Mixing Enables Soluble, Active Biomanufacturing of Difficult-to-Express and Aggregation-Prone Proteins",
  "type": "preprint",
  "updated_at": "2026-10-30T12:27:19Z",
  "url": "https://research.draconitic.com/paper/9rpsqxgq0s3f6",
  "venue": "bioRxiv",
  "versions": []
}