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Site‐Specific Protein Modification Using Lipoic Acid Ligase and Bis‐Aryl Hydrazone Formation
Author(s) -
Cohen Justin D.,
Zou Peng,
Ting Alice Y.
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100764
Subject(s) - chemistry , dna ligase , aldehyde , streptavidin , aryl , hydrazone , peptide , residue (chemistry) , lipoic acid , combinatorial chemistry , biochemistry , conjugate , stereochemistry , biotin , enzyme , organic chemistry , mathematical analysis , alkyl , mathematics , antioxidant , catalysis
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.