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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Expression testing of full-length human Rh membrane proteins in transiently transfected HEK293S GnTI cells.
Figure 2
Figure 3: Dilution of transfected HEK293S GnTI cells before drug selection.
Figure 4: Expression levels from stably transfected and clonal HEK293S GnTI cell lines as determined by western blotting.
Figure 5: Medium- and large-scale HEK293S GnTI cell cultures.

References

  1. 1

    Engelman, D.M. et al. Membrane protein folding: beyond the two-stage model. FEBS Lett. 555, 122–125 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Popot, J.L. & Engelman, D.M. Membrane protein folding and oligomerization: the two-stage model. Biochemistry 29, 4031–4037 (1990).

    CAS  Article  Google Scholar 

  3. 3

    Hessa, T. et al. Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377–381 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Rapoport, T.A., Jungnickel, B. & Kutay, U. Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu. Rev. Biochem. 65, 271–303 (1996).

    CAS  Article  Google Scholar 

  5. 5

    Wagner, S. et al. Consequences of membrane protein overexpression in Escherichia coli. Mol. Cell Proteomics 6, 1527–1550 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Wagner, S. et al. Tuning Escherichia coli for membrane protein overexpression. Proc. Natl. Acad. Sci. USA 105, 14371–14376 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Bowie, J.U. Solving the membrane protein folding problem. Nature 438, 581–589 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Klepsch, M.M., Persson, J.O. & de Gier, J.W. Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli. J. Mol. Biol. 407, 532–542 (2011).

    CAS  Article  Google Scholar 

  9. 9

    White, S.H. & Wimley, W.C. Membrane protein folding and stability: physical principles. Annu. Rev. Biophys. Biomol. Struct. 28, 319–365 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Dowhan, W. & Bogdanov, M. Lipid-dependent membrane protein topogenesis. Annu. Rev. Biochem. 78, 515–540 (2009).

    CAS  Article  Google Scholar 

  11. 11

    Spector, A.A. & Yorek, M.A. Membrane lipid composition and cellular function. J. Lipid Res. 26, 1015–1035 (1985).

    CAS  PubMed  Google Scholar 

  12. 12

    Guan, L., Smirnova, I.N., Verner, G., Nagamori, S. & Kaback, H.R. Manipulating phospholipids for crystallization of a membrane transport protein. Proc. Natl. Acad. Sci. USA 103, 1723–1726 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Long, S.B., Campbell, E.B. & Mackinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Tate, C.G. Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett. 504, 94–98 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Helenius, A. & Aebi, M. Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019–1049 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Kwong, P.D. et al. Probability analysis of variational crystallization and its application to gp120, the exterior envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1). J. Biol. Chem. 274, 4115–4123 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Aricescu, A.R., Lu, W. & Jones, E.Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D Biol. Crystallogr. 62, 1243–1250 (2006).

    Article  Google Scholar 

  18. 18

    Chang, V.T. et al. Glycoprotein structural genomics: solving the glycosylation problem. Structure 15, 267–273 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Lee, J.E., Fusco, M.L. & Ollmann Saphire, E. An efficient platform for screening expression and crystallization of glycoproteins produced in human cells. Nat. Protoc. 4, 592–604 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Reeves, P.J., Callewaert, N., Contreras, R. & Khorana, H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

    CAS  Article  Google Scholar 

  21. 21

    Gruswitz, F. et al. Function of human Rh based on structure of RhCG at 2.1 A. Proc. Natl. Acad. Sci. USA 107, 9638–9643 (2010).

    CAS  Article  Google Scholar 

  22. 22

    Standfuss, J. et al. Crystal structure of a thermally stable rhodopsin mutant. J. Mol. Biol. 372, 1179–1188 (2007).

    CAS  Article  Google Scholar 

  23. 23

    Chelikani, P., Reeves, P.J., Rajbhandary, U.L. & Khorana, H.G. The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line. Protein Sci. 15, 1433–1440 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Reeves, P.J., Thurmond, R.L. & Khorana, H.G. Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines. Proc. Natl. Acad. Sci. USA 93, 11487–11492 (1996).

    CAS  Article  Google Scholar 

  25. 25

    Takayama, H., Chelikani, P., Reeves, P.J., Zhang, S. & Khorana, H.G. High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81. PLoS ONE 3, e2314 (2008).

    Article  Google Scholar 

  26. 26

    Rosenbaum, D.M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Wurm, F. & Bernard, A. Large-scale transient expression in mammalian cells for recombinant protein production. Curr. Opin. Biotechnol. 10, 156–159 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Meissner, P. et al. Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol. Bioeng. 75, 197–203 (2001).

    CAS  Article  Google Scholar 

  29. 29

    Bailey, C.G., Tait, A.S. & Sunstrom, N.A. High-throughput clonal selection of recombinant CHO cells using a dominant selectable and amplifiable metallothionein-GFP fusion protein. Biotechnol. Bioeng. 80, 670–676 (2002).

    CAS  Article  Google Scholar 

  30. 30

    White, M.A., Clark, K.M., Grayhack, E.J. & Dumont, M.E. Characteristics affecting expression and solubilization of yeast membrane proteins. J. Mol. Biol. 365, 621–636 (2007).

    CAS  Article  Google Scholar 

  31. 31

    Lewinson, O., Lee, A.T. & Rees, D.C. The funnel approach to the precrystallization production of membrane proteins. J. Mol. Biol. 377, 62–73 (2008).

    CAS  Article  Google Scholar 

  32. 32

    Reeves, P.J., Kim, J.M. & Khorana, H.G. Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc. Natl. Acad. Sci. USA 99, 13413–13418 (2002).

    CAS  Article  Google Scholar 

  33. 33

    Schlaeger, E.J. The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties. J. Immunol. Methods 194, 191–199 (1996).

    CAS  Article  Google Scholar 

  34. 34

    Tharmalingam, T., Ghebeh, H., Wuerz, T. & Butler, M. Pluronic enhances the robustness and reduces the cell attachment of mammalian cells. Mol. Biotechnol. 39, 167–177 (2008).

    CAS  Article  Google Scholar 

  35. 35

    Falk, T. et al. Over-expression of the potassium channel Kir2.3 using the dopamine-1 receptor promoter selectively inhibits striatal neurons. Neuroscience 155, 114–127 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Lohse, M.J. Stable overexpression of human beta 2-adrenergic receptors in mammalian cells. Naunyn. Schmiedebergs Arch. Pharmacol. 345, 444–451 (1992).

    CAS  Article  Google Scholar 

  37. 37

    Gorman, C.M., Howard, B.H. & Reeves, R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 11, 7631–7648 (1983).

    CAS  Article  Google Scholar 

  38. 38

    Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    CAS  Article  Google Scholar 

  39. 39

    Newby, Z.E. et al. A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat. Protoc. 4, 619–637 (2009).

    CAS  Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Corresponding author

Correspondence to Robert M Stroud.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Full Sequence of pACMV-tetO. (DOC 27 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chaudhary, S., Pak, J., Gruswitz, F. et al. Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nat Protoc 7, 453–466 (2012). https://doi.org/10.1038/nprot.2011.453

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.