title: Labeling proteins on live mammalian cells using click chemistry.
abstract: We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Ivana
authors.identifier: 0000000266663486
authors.initials: I
authors.lastname: Nikić
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Jun Hee
authors.initials: JH
authors.lastname: Kang
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Gemma Estrada
authors.initials: GE
authors.lastname: Girona
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Iker Valle
authors.initials: IV
authors.lastname: Aramburu
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Lemke
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D057930:Click Chemistry / Q000295:instrumentation / Q000379:methods*; D018388:Codon, Terminator; D061565:Cycloaddition Reaction; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics
chemical_list: C569686:Alexa Fluor 647; D000480:Alkynes; D000596:Amino Acids; D001386:Azides; D002232:Carbocyanines; D018388:Codon, Terminator; D005456:Fluorescent Dyes; D011506:Proteins; C085321:cyanine dye 5; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin; D000604:Amino Acyl-tRNA Synthetases