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In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag

Nature Methods volume 2, pages 255–257 (2005)Cite this article

Abstract

The introduction of green fluorescent protein and its variants (GFPs) has allowed protein analysis at the level of the cell. Now, chemical methods are needed to label proteins in vivo with a wider variety of functionalities so that mechanistic questions about protein function in the complex cellular environment can be addressed. Here we demonstrate that trimethoprim derivatives can be used to selectively tag Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins in wild-type mammalian cells with minimal background and fast kinetics.

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Figure 1: General strategy of protein labeling and synthesis of fluorescent TMP.
Figure 2: Selective chemical labeling of subcellularly targeted eDHFR in wild-type CHO cells.
Figure 3: Selective chemical labeling of eDHFR fusion proteins in MEF cells.

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Acknowledgements

This research was supported by the National Institutes of Health (GM071754-01). V.W.C. is a recipient of a Beckman Young Investigator Award, a Burroughs Wellcome Fund New Investigator Award in the Toxicological Sciences, a Camille and Henry Dreyfus New Faculty Award and a National Science Foundation Career Award.

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Authors and Affiliations

  1. Department of Chemistry, Columbia University, 3,000 Broadway, New York, 10027, USA

    Lawrence W Miller & Virginia W Cornish

  2. Department of Biological Sciences, Columbia University, 3,000 Broadway, New York, 10027, USA

    Lawrence W Miller, Yunfei Cai & Michael P Sheetz

Authors
  1. Lawrence W Miller
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  2. Yunfei Cai
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  3. Michael P Sheetz
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  4. Virginia W Cornish
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Correspondence to Lawrence W Miller.

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Supplementary information

Supplementary Fig. 1

The affinity of TMP-fluorescein for eDHFR was determined by fluorescence polarization. (GIF 33 kb)

Supplementary Fig. 2

Confocal micrographs showing an MEF cell expressing a fusion of puromycin n-acetyl transferase to GFP. (JPG 16 kb)

Supplementary Fig. 3

MEF cells expressing puromycin n-acetyl transferase-eDHFR. (JPG 35 kb)

Supplementary Methods (PDF 199 kb)

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Miller, L., Cai, Y., Sheetz, M. et al. In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag. Nat Methods 2, 255–257 (2005). https://doi.org/10.1038/nmeth749

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