An Engineered Protein Tag for Multiprotein Labeling in Living Cells

Chem Biol. 2008 Feb;15(2):128-36. doi: 10.1016/j.chembiol.2008.01.007.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • CHO Cells
  • Cell Survival*
  • Cricetinae
  • Cricetulus
  • Cytosine / analogs & derivatives
  • Cytosine / metabolism
  • Fluorescent Dyes / chemical synthesis*
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism*
  • Models, Molecular
  • Mutation
  • O(6)-Methylguanine-DNA Methyltransferase / genetics
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism
  • Protein Conformation
  • Proteins / metabolism*
  • Staining and Labeling / methods*
  • Substrate Specificity

Substances

  • Fluorescent Dyes
  • Proteins
  • Cytosine
  • O(6)-Methylguanine-DNA Methyltransferase