title: An engineered protein tag for multiprotein labeling in living cells.
abstract: The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.
authors.affiliation: Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Gautier
authors.forename: Alexandre
authors.initials: A
authors.lastname: Juillerat
authors.forename: Christian
authors.initials: C
authors.lastname: Heinis
authors.forename: Ivan Reis
authors.initials: IR
authors.lastname: Corrêa
authors.forename: Maik
authors.initials: M
authors.lastname: Kindermann
authors.forename: Florent
authors.initials: F
authors.lastname: Beaufils
authors.forename: Kai
authors.initials: K
authors.lastname: Johnsson
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D003596:Cytosine; D019853:O(6)-Methylguanine-DNA Methyltransferase