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large family of structurally diverse and natural products produced by bacteria and fungi

3.5kdb. Ladder 0h 1h 2h 4h 6h 8h 10h 24h. Isolation & Characterization of Polyketide Synthase Thioesth er ase Domains Evelyn Walenta & Christopher N. Boddy* Department of Chemistry, Syracuse University Sy racuse, NY 13244.

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large family of structurally diverse and natural products produced by bacteria and fungi

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  1. 3.5kdb Ladder 0h 1h 2h 4h 6h 8h 10h 24h Isolation & Characterization of Polyketide Synthase Thioestherase DomainsEvelyn Walenta & Christopher N. Boddy*Department of Chemistry, Syracuse UniversitySyracuse, NY 13244 Polyketides are important clinical agents Amphotericin TE Protein Expression Project Goal: Characterize multiple TE Domains • Amphotericine, Nystatin, Avermectin, Oleandomycin TE domains were selected • BL21 (CDE3) were used for protein expression) • Time course for protein expression at 30°C shows 10h to be optimal • Amphotericin TE was purified by Ni-NTA chromatography • 4 ml of 22 µM high purity Amphotericin TE was obtained from 0.5 l of culture Amphotericin Nystatin Why? • TE domains were sequenced • bacterial strains were commercially available • Polyketide products were commercially available Avermctin Oleandomycin ~6.5 ~5.5 • large family of structurally diverse and natural products produced by bacteria and fungi Generation of TE expression vector Activity of Amphotericin TE Polyketides are made in an assembly-line fashion • Amphotericin TE does not catalyze lactone hydrolosys producing organisms isolate genomic DNA grow culture TE domain PCR LCMS analysis shows no hydrolysis activity clone into expression vecoor ~950 • Thioester Domain can hydrolyze simple Thioesters TE domain Expression vector for Amphotericin TE was generated PCR products for all 4 TE domains were generated • Enormous multifunktional enzymes • Growing polyketide is covalently attached to enzyme • Complex mulecules are made from simple starting materials • Different organisation of modules gives different polyketides LCMS analysis shows hydrolysis activity Acknowledgements Release of Polyketide from enzyme in crucial Ben Lundgren (Syracuse University) Mike Henry (Syracuse University) The Boddy Lab (Syracuse University) Syracuse University University of Technology (Graz) cyclorelease - HSR R: ACP or N-Acetulcysteamine • Thioester domain catalyzes the release of the polyketide via cyclization

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