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Site-Specific Protein Modification Using Lipoic Acid Ligase and Bis-Aryl Hydrazone Formation

Authors

  • Dr. Justin D. Cohen,

    1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA, 02139 (USA)
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  • Peng Zou,

    1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA, 02139 (USA)
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  • Prof. Alice Y. Ting

    Corresponding author
    1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA, 02139 (USA)
    • Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA, 02139 (USA)
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Abstract

A screen of Trp37 mutants of Escherichia coli lipoic acid ligase (LplA) revealed enzymes capable of ligating an aryl-aldehyde or aryl-hydrazine substrate to LplA's 13-residue acceptor peptide. Once site-specifically attached to recombinant proteins fused to this peptide, aryl-aldehydes could be chemoselectively derivatized with hydrazine-probe conjugates, and aryl-hydrazines could be derivatized in an analogous manner with aldehyde-probe conjugates. Such two-step labeling was demonstrated for AlexaFluor568 targeting to monovalent streptavidin in vitro, and to neurexin-1β on the surface of living mammalian cells. To further highlight this technique, we labeled the low-density lipoprotein receptor on the surface of live cells with fluorescent phycoerythrin protein to allow single-molecule imaging and tracking over time.

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