Expression of Fluorescent Cyclotides using Protein Trans-Splicing for Easy Monitoring of Cyclotide–Protein Interactions

Authors

  • Dr. Krishnappa Jagadish,

    1. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA)
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  • Dr. Radhika Borra,

    1. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA)
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  • Vanessa Lacey,

    1. Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, CA 92037 (USA)
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  • Subhabrata Majumder,

    1. Department of Chemistry, State University of New York, Albany, NY 12222 (USA)
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  • Dr. Alexander Shekhtman,

    1. Department of Chemistry, State University of New York, Albany, NY 12222 (USA)
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  • Dr. Lei Wang,

    1. Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, CA 92037 (USA)
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  • Dr. Julio A. Camarero

    Corresponding author
    1. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA)
    2. Department of Chemistry, University of Southern California, Los Angeles, CA 90033 (USA)
    • Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (USA)

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  • This work was supported by National Institutes of Health Research Grants R01-GM090323 (J.A.C.), R01-GM085006 (A.S.), DP2-OD004744 (L.W.) and by the Department of Defense Congressionally Directed Medical Research Program Grant PC09305 (J.A.C.). We would also like to thank Sam Ulin (Salk Institute) for his contribution in the preparation of plasmid pERAzi.

Abstract

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Fremdling im Ring: Die Expression von Cyclotiden mit nichtnatürlichen Aminosäuren (grauer Kreis im Schema) in lebenden Bakterienzellen gelingt mithilfe eines hoch effizienten getrennten Inteins in Kombination mit Nonsense-Codon-Suppressor-tRNA-Technik. p-Azidophenylalanin-haltige Cyclotide können in Bakterien exprimiert und leicht über kupferfreie Klick-Reaktionen markiert werden, um Cyclotid-Protein-Wechselwirkungen nachzuvollziehen.

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