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Optimization of membrane protein overexpression and purification using GFP fusions

Nature Methods volume 3, pages 303–313 (2006)Cite this article

  • 1,4-dithiothreitol (DTT; Sigma)

  • Buffer A: phosphate-buffered saline (PBS) with 0.1% n-dodecyl-β-D-maltopyranoside (DDM; or other detergent at 5× critical micellar concentration; see Supplementary Table 1 online)

  • Buffer B: Buffer A with 500 mM imidazole

  • Deoxyribonuclease I from bovine pancreas Type IV lyophilized powder (Sigma)

  • E. coli BL21(DE3)–derived host strains (see Supplementary Table 2 online)

  • Ethylenediaminetetraacetic acid (EDTA; Sigma)

  • Purified GFP (Supplementary Methods online)

  • Solubilization buffer (SB): 200 mM Tris-HCl (pH 8.8), 20% Glycerol, 5 mM EDTA (pH 8.0), 0.02% bromphenol blue, make aliquots of 700 μl and keep at −20 °C. Before use, add 200 μl 20% SDS and 100 μl 0.5 M DDT

  • Tobacco etch virus (TEV) protease, His-tagged (see Supplementary Data online)

1,4-dithiothreitol (DTT; Sigma)

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Figure 1
Figure 2: Monitoring overexpression of membrane protein GFP fusions using whole-cell and in-gel fluorescence.
Figure 3: Examples of method application.

References

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Acknowledgements

Research in the lab of J.W.d.G. is supported by the Swedish Research Council, the European Molecular Biology Organization Young Investigator Programme (EMBO YIP) and the Marianne and Marcus Wallenberg Foundation. D.D. was a recipient of European Science Foundation (ESF) and EMBO short-term fellowships. M.L. was a recipient of a fellowship from the Swiss National Science Foundation. Research in the laboratory of E.K. is supported by the Medical Research Council (MRC) and the EMBO YIP. D.J.S. was supported by a long-term fellowship of the Human Frontier Science Program Organization. Gunnar von Heijne is acknowledged for his continuous support.

Author information

Author notes

  1. David Drew and Mirjam Lerch: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91, Sweden

    David Drew, Mirjam Lerch & Jan-Willem de Gier

  2. MRC Dunn Human Nutrition Unit, Hills Road, Cambridge, CB2 2XY, United Kingdom

    Edmund Kunji

  3. Department of Biochemistry, University of Groningen, Nyenborg 4, Groningen, 9747 AG, The Netherlands

    Dirk-Jan Slotboom

Authors
  1. David Drew
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  2. Mirjam Lerch
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  3. Edmund Kunji
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  4. Dirk-Jan Slotboom
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  5. Jan-Willem de Gier
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Corresponding author

Correspondence to Jan-Willem de Gier.

Supplementary information

Supplementary Fig. 1

Expression vectors. (PDF 410 kb)

Supplementary Fig. 2

GFP fluorescence is dampened in whole-cells and non-detergent solubilized membranes. (PDF 646 kb)

Supplementary Table 1

Detergents. (DOC 57 kb)

Supplementary Table 2

BL21 derived strains. (DOC 53 kb)

Supplementary Methods (DOC 55 kb)

Supplementary Data (DOC 43 kb)

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Drew, D., Lerch, M., Kunji, E. et al. Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 3, 303–313 (2006). https://doi.org/10.1038/nmeth0406-303

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