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

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

Author information

1
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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.

PMID:
22492621
PMCID:
PMC4758125
DOI:
10.1002/cbic.201100764
[Indexed for MEDLINE]
Free PMC Article

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