title: Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation.
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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Justin D
authors.initials: JD
authors.lastname: Cohen
authors.forename: Peng
authors.initials: P
authors.lastname: Zou
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057054:Molecular Imaging; D010799:Phycoerythrin / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D008063:Thioctic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D010799:Phycoerythrin; D008063:Thioctic Acid; D019809:Streptavidin; D008025:Ligases