Protocol | Published:

Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells

Nature Protocols volume 7, pages 453466 (2012) | Download Citation

Abstract

X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI), and was recently used in our 2.1-Å X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of 0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8–10 weeks.

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Acknowledgements

This work was supported by National Institutes of Health/National Institute of General Medical Sciences grants P50 GM73210, U54 GM094625 and R37 GM24485.

Author information

Author notes

    • Sarika Chaudhary
    •  & John E Pak

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA.

    • Sarika Chaudhary
    • , John E Pak
    • , Franz Gruswitz
    •  & Robert M Stroud
  2. Center for the Structure of Membrane Proteins, University of California, San Francisco, California, USA.

    • Sarika Chaudhary
    • , John E Pak
    • , Franz Gruswitz
    •  & Robert M Stroud
  3. Membrane Protein Expression Center, University of California, San Francisco, California, USA.

    • Sarika Chaudhary
    • , John E Pak
    • , Franz Gruswitz
    •  & Robert M Stroud
  4. Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India.

    • Vinay Sharma

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Contributions

S.C., F.G. and R.M.S. designed the experiments. S.C. and F.G. performed the experiments. S.C., J.E.P., F.G. and R.M.S. analyzed the data. V.S. and R.M.S. supervised personnel. S.C., J.E.P. and R.M.S. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Robert M Stroud.

Supplementary information

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  1. 1.

    Supplementary Fig. 1

    Full Sequence of pACMV-tetO.

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DOI

https://doi.org/10.1038/nprot.2011.453

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