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Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

Nature Protocols volume 9, pages 2574–2585 (2014)Cite this article

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Abstract

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8–tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI− (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4–6 weeks.

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Figure 1: Flowchart of the overexpression of membrane proteins in HEK293S GnTI cells.
Figure 2: Map of BacMam expression vector.
Figure 3: Screening constructs by small-scale transient transfection in HEK293S GnTI− cells and FSEC; 1 μg of N-terminal EGFP-tagged full-length cASIC1 or cASIC1 Δ463 (cASIC1, truncated 64 residues from the carboxy termini) subcloned into pEG BacMam was transfected separately into HEK293S GnTI− cells, as described in the text.
Figure 4: Time course of expression of cASIC1 and GluCl in HEK293S GnTI− cells.
Figure 5: Effect of histone deacetylase inhibitors on the expression of cASIC1 and GluCl in HEK293S GnTI− cells.
Figure 6: Comparison of expression time course in HEK293S GnTI− cells and Sf9 cells.

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Acknowledgements

We thank members of the Gouaux laboratory for helpful discussions. We are grateful to D. Goodman and G. Westbrook for encouragement and L. Vaskalis for assistance with figures. This work was supported by an Oregon Health and Science University Brain Institute Graduate Student Fellowship for Research on the Neurobiology of Disease (C.-H.L.), by a F32 Postdoctoral National Research Service Award (NRSA) from the US National Institute of Mental Health (NIMH) (K.H.W.), by a postdoctoral fellowship (Forschungsstipendium AL 1725-1/1) from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (T.A.), by a F32 Postdoctoral NRSA from the US National Institute of General Medical Sciences (NIGMS) (D.P.C.), by the US National Institutes of Health (NIH) (E.G.) and by the Vollum Institute. E.G. is an investigator with the Howard Hughes Medical Institute.

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Author notes

  1. Thorsten Althoff

    Present address: Present address: Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.,

  2. April Goehring and Chia-Hsueh Lee: These authors contributed equally to this work.

Authors and Affiliations

  1. Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA

    April Goehring, Chia-Hsueh Lee, Kevin H Wang, Jennifer Carlisle Michel, Derek P Claxton, Isabelle Baconguis, Thorsten Althoff & Eric Gouaux

  2. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    April Goehring, Jennifer Carlisle Michel, K Christopher Garcia & Eric Gouaux

  3. Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, California, USA

    Suzanne Fischer & K Christopher Garcia

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Contributions

A.G. screened and optimized expression conditions for cASIC1 and GluCl. A.G., C.-H.L., K.H.W., J.C.M. and D.P.C. optimized the cell growth and virus amplification conditions. C.-H.L. designed the BacMam construct and performed initial characterization of the BacMam construct. I.B. and T.A. cloned and optimized the cASIC1 and GluCl pEG BacMam constructs, respectively. K.C.G. and S.F. provided the pVLAD construct, incubator configuration and consultations to optimize cell growth during the early stages of the project. All authors wrote and edited the manuscript.

Corresponding author

Correspondence to Eric Gouaux.

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Goehring, A., Lee, CH., Wang, K. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat Protoc 9, 2574–2585 (2014). https://doi.org/10.1038/nprot.2014.173

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