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. 2002 Dec;3(6):511–517. doi: 10.1002/cfg.218

Heterologous Expression and Purification Systems for Structural Proteomics of Mammalian Membrane Proteins

Isabelle Mus-Veteau 1,✉
PMCID: PMC2448422  PMID: 18629259

Abstract

Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural proteomics.

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Selected References

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  1. Altmann F., Staudacher E., Wilson I. B., März L. Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj J. 1999 Feb;16(2):109–123. doi: 10.1023/a:1026488408951. [DOI] [PubMed] [Google Scholar]
  2. Bill R. M. Yeast--a panacea for the structure-function analysis of membrane proteins? Curr Genet. 2001 Oct;40(3):157–171. doi: 10.1007/s002940100252. [DOI] [PubMed] [Google Scholar]
  3. Bouveret E., Rigaut G., Shevchenko A., Wilm M., Séraphin B. A Sm-like protein complex that participates in mRNA degradation. EMBO J. 2000 Apr 3;19(7):1661–1671. doi: 10.1093/emboj/19.7.1661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brower V. Proteomics: biology in the post-genomic era. Companies all over the world rush to lead the way in the new post-genomics race. EMBO Rep. 2001 Jul;2(7):558–560. doi: 10.1093/embo-reports/kve144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cereghino J. L., Cregg J. M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45–66. doi: 10.1111/j.1574-6976.2000.tb00532.x. [DOI] [PubMed] [Google Scholar]
  6. Chang G., Roth C. B. Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science. 2001 Sep 7;293(5536):1793–1800. doi: 10.1126/science.293.5536.1793. [DOI] [PubMed] [Google Scholar]
  7. Christendat D., Yee A., Dharamsi A., Kluger Y., Gerstein M., Arrowsmith C. H., Edwards A. M. Structural proteomics: prospects for high throughput sample preparation. Prog Biophys Mol Biol. 2000;73(5):339–345. doi: 10.1016/s0079-6107(00)00010-9. [DOI] [PubMed] [Google Scholar]
  8. Clayton Andrew H. A., Sawyer William H. Site-specific tryptophan fluorescence spectroscopy as a probe of membrane peptide structure and dynamics. Eur Biophys J. 2002 Mar;31(1):9–13. doi: 10.1007/s002490100182. [DOI] [PubMed] [Google Scholar]
  9. Cordat E., Leblanc G., Mus-Veteau I. Evidence for a role of helix IV in connecting cation- and sugar-binding sites of Escherichia coli melibiose permease. Biochemistry. 2000 Apr 18;39(15):4493–4499. doi: 10.1021/bi991852i. [DOI] [PubMed] [Google Scholar]
  10. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  11. Drew D. E., von Heijne G., Nordlund P., de Gier J. W. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett. 2001 Oct 26;507(2):220–224. doi: 10.1016/s0014-5793(01)02980-5. [DOI] [PubMed] [Google Scholar]
  12. Dutzler Raimund, Campbell Ernest B., Cadene Martine, Chait Brian T., MacKinnon Roderick. X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature. 2002 Jan 17;415(6869):287–294. doi: 10.1038/415287a. [DOI] [PubMed] [Google Scholar]
  13. Figler R. A., Omote H., Nakamoto R. K., Al-Shawi M. K. Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. Arch Biochem Biophys. 2000 Apr 1;376(1):34–46. doi: 10.1006/abbi.2000.1712. [DOI] [PubMed] [Google Scholar]
  14. Hunte C. Insights from the structure of the yeast cytochrome bc1 complex: crystallization of membrane proteins with antibody fragments. FEBS Lett. 2001 Aug 31;504(3):126–132. doi: 10.1016/s0014-5793(01)02744-2. [DOI] [PubMed] [Google Scholar]
  15. Iwata S., Lee J. W., Okada K., Lee J. K., Iwata M., Rasmussen B., Link T. A., Ramaswamy S., Jap B. K. Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science. 1998 Jul 3;281(5373):64–71. doi: 10.1126/science.281.5373.64. [DOI] [PubMed] [Google Scholar]
  16. Iwata S., Ostermeier C., Ludwig B., Michel H. Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature. 1995 Aug 24;376(6542):660–669. doi: 10.1038/376660a0. [DOI] [PubMed] [Google Scholar]
  17. Julien M., Kajiji S., Kaback R. H., Gros P. Simple purification of highly active biotinylated P-glycoprotein: enantiomer-specific modulation of drug-stimulated ATPase activity. Biochemistry. 2000 Jan 11;39(1):75–85. doi: 10.1021/bi991726e. [DOI] [PubMed] [Google Scholar]
  18. Kempf Juliette, Snook Laelie A., Vonesch Jean Luc, Dahms Tanya E. S., Pattus Franc, Massotte Dominique. Expression of the human mu opioid receptor in a stable Sf9 cell line. J Biotechnol. 2002 May 9;95(2):181–187. doi: 10.1016/s0168-1656(02)00008-1. [DOI] [PubMed] [Google Scholar]
  19. Kolbe M., Besir H., Essen L. O., Oesterhelt D. Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution. Science. 2000 May 26;288(5470):1390–1396. doi: 10.1126/science.288.5470.1390. [DOI] [PubMed] [Google Scholar]
  20. Lee G. M., Kim E. J., Kim N. S., Yoon S. K., Ahn Y. H., Song J. Y. Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design. J Biotechnol. 1999 Apr 15;69(2-3):85–93. doi: 10.1016/s0168-1656(99)00004-8. [DOI] [PubMed] [Google Scholar]
  21. Lenoir Guillaume, Menguy Thierry, Corre Fabienne, Montigny Cédric, Pedersen Per A., Thinès Denyse, le Maire Marc, Falson Pierre. Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. Biochim Biophys Acta. 2002 Feb 18;1560(1-2):67–83. doi: 10.1016/s0005-2736(01)00458-8. [DOI] [PubMed] [Google Scholar]
  22. Levy D., Chami M., Rigaud J. L. Two-dimensional crystallization of membrane proteins: the lipid layer strategy. FEBS Lett. 2001 Aug 31;504(3):187–193. doi: 10.1016/s0014-5793(01)02748-x. [DOI] [PubMed] [Google Scholar]
  23. Locher Kaspar P., Lee Allen T., Rees Douglas C. The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science. 2002 May 10;296(5570):1091–1098. doi: 10.1126/science.1071142. [DOI] [PubMed] [Google Scholar]
  24. Luecke H., Schobert B., Lanyi J. K., Spudich E. N., Spudich J. L. Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction. Science. 2001 Jul 12;293(5534):1499–1503. doi: 10.1126/science.1062977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miroux B., Walker J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 1996 Jul 19;260(3):289–298. doi: 10.1006/jmbi.1996.0399. [DOI] [PubMed] [Google Scholar]
  26. Murata K., Mitsuoka K., Hirai T., Walz T., Agre P., Heymann J. B., Engel A., Fujiyoshi Y. Structural determinants of water permeation through aquaporin-1. Nature. 2000 Oct 5;407(6804):599–605. doi: 10.1038/35036519. [DOI] [PubMed] [Google Scholar]
  27. Nollert P., Qiu H., Caffrey M., Rosenbusch J. P., Landau E. M. Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases. FEBS Lett. 2001 Aug 31;504(3):179–186. doi: 10.1016/s0014-5793(01)02747-8. [DOI] [PubMed] [Google Scholar]
  28. Palczewski K., Kumasaka T., Hori T., Behnke C. A., Motoshima H., Fox B. A., Le Trong I., Teller D. C., Okada T., Stenkamp R. E. Crystal structure of rhodopsin: A G protein-coupled receptor. Science. 2000 Aug 4;289(5480):739–745. doi: 10.1126/science.289.5480.739. [DOI] [PubMed] [Google Scholar]
  29. Pebay-Peyroula E., Rummel G., Rosenbusch J. P., Landau E. M. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science. 1997 Sep 12;277(5332):1676–1681. doi: 10.1126/science.277.5332.1676. [DOI] [PubMed] [Google Scholar]
  30. Quick Matthias, Wright Ernest M. Employing Escherichia coli to functionally express, purify, and characterize a human transporter. Proc Natl Acad Sci U S A. 2002 Jun 19;99(13):8597–8601. doi: 10.1073/pnas.132266599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rigaut G., Shevchenko A., Rutz B., Wilm M., Mann M., Séraphin B. A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol. 1999 Oct;17(10):1030–1032. doi: 10.1038/13732. [DOI] [PubMed] [Google Scholar]
  32. Stahlberg H., Fotiadis D., Scheuring S., Rémigy H., Braun T., Mitsuoka K., Fujiyoshi Y., Engel A. Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins. FEBS Lett. 2001 Aug 31;504(3):166–172. doi: 10.1016/s0014-5793(01)02746-6. [DOI] [PubMed] [Google Scholar]
  33. Tate C. G. Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett. 2001 Aug 31;504(3):94–98. doi: 10.1016/s0014-5793(01)02711-9. [DOI] [PubMed] [Google Scholar]
  34. Theis S., Döring F., Daniel H. Expression of the myc/His-tagged human peptide transporter hPEPT1 in yeast for protein purification and functional analysis. Protein Expr Purif. 2001 Aug;22(3):436–442. doi: 10.1006/prep.2001.1455. [DOI] [PubMed] [Google Scholar]
  35. Toyoshima C., Nakasako M., Nomura H., Ogawa H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature. 2000 Jun 8;405(6787):647–655. doi: 10.1038/35015017. [DOI] [PubMed] [Google Scholar]
  36. Tsukihara T., Aoyama H., Yamashita E., Tomizaki T., Yamaguchi H., Shinzawa-Itoh K., Nakashima R., Yaono R., Yoshikawa S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science. 1996 May 24;272(5265):1136–1144. doi: 10.1126/science.272.5265.1136. [DOI] [PubMed] [Google Scholar]
  37. Urbatsch I. L., Wilke-Mounts S., Gimi K., Senior A. E. Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells. Arch Biochem Biophys. 2001 Apr 1;388(1):171–177. doi: 10.1006/abbi.2001.2299. [DOI] [PubMed] [Google Scholar]
  38. Weiss H. M., Haase W., Michel H., Reiländer H. Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human beta2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris. Biochem J. 1998 Mar 15;330(Pt 3):1137–1147. doi: 10.1042/bj3301137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yelin R., Schuldiner S. Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding. Biochim Biophys Acta. 2001 Feb 9;1510(1-2):426–441. doi: 10.1016/s0005-2736(00)00374-6. [DOI] [PubMed] [Google Scholar]
  40. Zaworski P. G., Evans D. L., Lahti R. A., Gill G. S. Growth of Chinese hamster ovary (CHO) cells expressing the 5-HT2 serotonin receptor in suspension culture: an efficient method for large-scale acquisition of membrane protein for drug evaluation. J Neurosci Methods. 1995 Feb;56(2):169–175. doi: 10.1016/0165-0270(94)00119-2. [DOI] [PubMed] [Google Scholar]
  41. Zhou Y., Morais-Cabral J. H., Kaufman A., MacKinnon R. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. Nature. 2001 Nov 1;414(6859):43–48. doi: 10.1038/35102009. [DOI] [PubMed] [Google Scholar]

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