Protocol | Published:

Optimization of membrane protein overexpression and purification using GFP fusions

Nature Methods volume 3, pages 303313 (2006) | Download Citation

Subjects

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , & Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett. 507, 220–224 (2001).

  2. 2.

    et al. Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis. Proc. Natl. Acad. Sci. USA 99, 2690–2695 (2002).

  3. 3.

    et al. A scalable, GFP-based pipeline for membrane protein overexpression screening and purification. Protein Sci. 14, 2011–2017 (2005).

  4. 4.

    , , , & Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

  5. 5.

    & Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 7, 1029–1038 (1998).

  6. 6.

    , , , & Experimentally constrained topology models for 51,208 bacterial inner membrane proteins. J. Mol. Biol. 352, 489–494 (2005).

  7. 7.

    et al. Global topology analysis of the Escherichia coli inner membrane proteome. Science 308, 1321–1323 (2005).

  8. 8.

    et al. Experimentally based topology models for E. coli inner membrane proteins. Protein Sci. 13, 937–945 (2004).

  9. 9.

    et al. Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim. Biophys. Acta 1610, 23–36 (2003).

  10. 10.

    & Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase. Microb. Cell Fact. 4, 3 (2005).

  11. 11.

    , , , & Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J. Bacteriol. 182, 4068–4076 (2000).

  12. 12.

    , , , & Green fluorescent protein as a reporter of human mu-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris. J. Biotechnol. 99, 23–39 (2002).

  13. 13.

    , , , & Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301, 616–620 (2003).

Download references

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

    • David Drew
    •  & Mirjam Lerch

    These authors contributed equally to this work.

Affiliations

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

    • David Drew
    • , Mirjam Lerch
    •  & Jan-Willem de Gier
  2. MRC Dunn Human Nutrition Unit, Hills Road, CB2 2XY Cambridge, United Kingdom.

    • Edmund Kunji
  3. Department of Biochemistry, University of Groningen, Nyenborg 4, 9747 AG Groningen, the Netherlands.

    • Dirk-Jan Slotboom

Authors

  1. Search for David Drew in:

  2. Search for Mirjam Lerch in:

  3. Search for Edmund Kunji in:

  4. Search for Dirk-Jan Slotboom in:

  5. Search for Jan-Willem de Gier in:

Corresponding author

Correspondence to Jan-Willem de Gier.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Expression vectors.

  2. 2.

    Supplementary Fig. 2

    GFP fluorescence is dampened in whole-cells and non-detergent solubilized membranes.

Word documents

  1. 1.

    Supplementary Table 1

    Detergents.

  2. 2.

    Supplementary Table 2

    BL21 derived strains.

  3. 3.

    Supplementary Methods

  4. 4.

    Supplementary Data

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nmeth0406-303

Further reading