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Reconstitutionof Human Cytochrome P450 Activity usinga Leishmania Cell-Free Protein Expression System 利用利什曼原虫无细胞蛋白表达系统重组人细胞色素P450活性

Figshare Pub Date : 2025-09-15 DOI: 10.1021/acssynbio.5c00443.s001

WayneA. Johnston (2919251), Raine E. S. Thomson (14002736), Juan Alfaro-Palma (17844168), Robert E. Speight (8667261), Kirill Alexandrov (234705), Elizabeth M. J. Gillam (14002745), James B. Y. Behrendorff (22246448)

{"title":"Reconstitutionof Human Cytochrome P450 Activity usinga <i>Leishmania</i> Cell-Free Protein Expression System","authors":"Wayne\u0000A. Johnston (2919251), Raine E. S. Thomson (14002736), Juan Alfaro-Palma (17844168), Robert E. Speight (8667261), Kirill Alexandrov (234705), Elizabeth M. J. Gillam (14002745), James B. Y. Behrendorff (22246448)","doi":"10.1021/acssynbio.5c00443.s001","DOIUrl":"https://doi.org/10.1021/acssynbio.5c00443.s001","url":null,"abstract":"Cytochrome P450 enzymes (P450s) are ubiquitous in drug metabolism and natural product biosynthesis. Studying eukaryotic P450s has been limited by their dependence on membrane association and the requirement for partner reductases. Here, we demonstrate cell-free synthesis and assay of human P450s 3A4 and 2D6 using Leishmania tarentolae translational extract. These P450s were coexpressed with various NADPH-cytochrome P450 reductases (CPRs), and activity was assayed directly using unpurified reactions. P450s 3A4 and 2D6 showed distinct preferences in reductase coupling: P450 3A4 activity was greatest when coupled to the human CPR, whereas P450 2D6 performed better when coexpressed with CPRs from Arabidopsis thaliana. Inhibition assays with chloramphenicol, terbinafine, and erythromycin yielded results consistent with known P450-drug interactions. We conclude that <i>Leishmania</i>-based cell-free protein synthesis resolves previous challenges in eukaryotic P450 expression, allowing for rapid and convenient functional studies of unmodified eukaryotic P450 systems and offering a practical tool for drug metabolism studies and biocatalyst discovery.","PeriodicalId":521600,"journal":{"name":"Figshare","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147882239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

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