Journal
CHEMISTRY & BIOLOGY
Volume 15, Issue 2, Pages 128-136Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2008.01.007
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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 O-6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O-6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O-2-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.
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