Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation

Chembiochem. 2012 Apr 16;13(6):888-94. doi: 10.1002/cbic.201100764.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Cross-Linking Reagents / chemistry
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Ligases / chemistry*
  • Ligases / genetics
  • Ligases / metabolism
  • Molecular Imaging
  • Phycoerythrin / chemistry
  • Streptavidin / chemistry
  • Streptavidin / genetics
  • Streptavidin / metabolism
  • Substrate Specificity
  • Thioctic Acid / chemistry
  • Thioctic Acid / genetics
  • Thioctic Acid / metabolism*
  • Transfection


  • Cross-Linking Reagents
  • Escherichia coli Proteins
  • Phycoerythrin
  • Thioctic Acid
  • Streptavidin
  • Ligases