Labeling of fusion proteins with synthetic fluorophores in live cells

doi:10.1073/pnas.0401923101

Labeling of fusion proteins with synthetic fluorophores in live cells

Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland

Edited by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved May 24, 2004 (received for review March 18, 2004)

Abstract

A general approach for the sequential labeling of fusion proteins of O⁶-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.

Footnotes

↵ † To whom correspondence should be addressed. E-mail: kai.johnsson{at}epfl.ch.
↵ * A.K. and H.P. contributed equally to this work.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: AGT, O⁶-alkylguanine-DNA alkyltransferase; BG, O⁶-benzylguanine; FRET, fluorescence resonance energy transfer; OG, Oregon green; TMR, tetramethylrhodamine; SF, SNARF-1; β-Gal, β-galactosidase; tsVSVG, thermosensitive glycoprotein of vesicular stomatitis virus; NK₁, neurokinin-1; EGFP, enhanced GFP; NLS₃, three consecutive simian virus 40 nuclear localization sequences; CHO, Chinese hamster ovary; AF, diacetylated fluorescein; SP-rho, NK₁-specific ligand substance P.