An engineered protein tag for multiprotein labeling in living cells.
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.
Gautier
Arnaud
A
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Juillerat
Alexandre
A
Heinis
Christian
C
Corrêa
Ivan Reis
IR
Kindermann
Maik
M
Beaufils
Florent
F
Johnsson
Kai
K
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
D005456:Fluorescent Dyes; D011506:Proteins; D003596:Cytosine; D019853:O(6)-Methylguanine-DNA Methyltransferase