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Sortase-Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein

Authors

  • Yizhi Qi,

    1. Department of Biomedical Engineering, Duke University, Durham, NC, USA
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  • Miriam Amiram,

    1. Department of Biomedical Engineering, Duke University, Durham, NC, USA
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    • Present address: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06510, USA

  • Weiping Gao,

    1. Department of Biomedical Engineering, Duke University, Durham, NC, USA
    2. Center for Biologically Inspired Materials and Materials Systems, Duke University, Durham, NC, USA
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    • Present address: Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, P. R. China

  • Dewey G. McCafferty,

    1. Department of Chemistry, Duke University, Durham, NC, USA
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  • Ashutosh Chilkoti

    Corresponding author
    1. Center for Biologically Inspired Materials and Materials Systems, Duke University, Durham, NC, USA
    • Department of Biomedical Engineering, Duke University, Durham, NC, USA
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E-mail: chilkoti@duke.edu

Abstract

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Conventional methods for synthesizing protein/peptide–polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide–polymer conjugates.

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