title: Site-specific protein labeling using PRIME and chelation-assisted click chemistry.
abstract: This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches a picolyl azide (pAz) derivative to a 13-aa recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing pAz are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize pAz to perform PRIME labeling and to achieve CuAAC derivatization of pAz on live cells, fixed cells and purified proteins. Reagent preparations, including synthesis of pAz probes and expression of LplA, take 12 d, whereas the procedure for performing site-specific pAz ligation and CuAAC on cells or on purified proteins takes 40 min-3 h.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Chayasith
authors.initials: C
authors.lastname: Uttamapinant
authors.forename: Mateo I
authors.initials: MI
authors.lastname: Sanchez
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Liu
authors.forename: Jennifer Z
authors.initials: JZ
authors.lastname: Yao
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002614:Chelating Agents; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008025:Ligases / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000480:Alkynes; D001386:Azides; D002614:Chelating Agents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011506:Proteins; C490345:lplA protein, E coli; D003300:Copper; D008025:Ligases