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Expression and Purification of Membrane Proteins in Saccharomyces cerevisiae

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Expression, Purification, and Structural Biology of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2127))

Abstract

Saccharomyces cerevisiae is one of the most popular expression systems for eukaryotic membrane proteins. Here, we describe protocols for the expression and purification of mitochondrial membrane proteins developed in our laboratory during the last 15 years. To optimize their expression in a functional form, different promoter systems as well as codon-optimization and complementation strategies were established. Purification approaches were developed which remove the membrane protein from the affinity column by specific proteolytic cleavage rather than by elution. This strategy has several important advantages, most notably improving the purity of the sample, as contaminants stay bound to the column, thus eliminating the need for a secondary purification step, such as size exclusion chromatography. This strategy also avoids dilution of the sample, which would occur as a consequence of elution, precluding the need for concentration steps, and thus preventing detergent concentration.

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References

  1. Routledge SJ, Mikaliunaite L, Patel A, Clare M, Cartwright SP, Bawa Z, Wilks MDB, Low F, Hardy D, Rothnie AJ, Bill RM (2016) The synthesis of recombinant membrane proteins in yeast for structural studies. Methods 95:26–37. https://doi.org/10.1016/j.ymeth.2015.09.027

    Article  CAS  PubMed  Google Scholar 

  2. King MS, Boes C, Kunji ER (2015) Membrane protein expression in Lactococcus lactis. Methods Enzymol 556:77–97. https://doi.org/10.1016/bs.mie.2014.12.009

    Article  CAS  PubMed  Google Scholar 

  3. Contreras-Gomez A, Sanchez-Miron A, Garcia-Camacho F, Molina-Grima E, Chisti Y (2014) Protein production using the baculovirus-insect cell expression system. Biotechnol Prog 30(1):1–18. https://doi.org/10.1002/btpr.1842

    Article  CAS  PubMed  Google Scholar 

  4. Goehring A, Lee C-H, Wang KH, Michel JC, Claxton DP, Baconguis I, Althoff T, Fischer S, Garcia KC, Gouaux E (2014) Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat Prot 9(11):2574–2585. https://doi.org/10.1038/nprot.2014.173

    Article  CAS  Google Scholar 

  5. King MS, Kerr M, Crichton PG, Springett R, Kunji ER (2016) Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier. Biochim Biophys Acta 1857:14–22. https://doi.org/10.1016/j.bbabio.2015.09.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ruprecht JJ, Hellawell AM, Harding M, Crichton PG, Mccoy AJ, Kunji ERS (2014) Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism. Proc Natl Acad Sci U S A 111(4):E426–E434. https://doi.org/10.1073/Pnas.1320692111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ruprecht JJ, King MS, Zogg T, Aleksandrova AA, Pardon E, Crichton PG, Steyaert J, Kunji ERS (2019) The molecular mechanism of transport by the mitochondrial ADP/ATP carrier. Cell 176(3):435–447. https://doi.org/10.1016/j.cell.2018.11.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bamber L, Harding M, Butler PJG, Kunji ERS (2006) Yeast mitochondrial ADP/ATP carriers are monomeric in detergents. Proc Natl Acad Sci U S A 103(44):16224–16229. https://doi.org/10.1073/Pnas.0607640103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bamber L, Harding M, Monné M, Slotboom DJ, Kunji ERS (2007) The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes. Proc Natl Acad Sci U S A 104(26):10830–10834

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bamber L, Slotboom DJ, Kunji ERS (2007) Yeast mitochondrial ADP/ATP carriers are monomeric in detergents as demonstrated by differential affinity purification. J Mol Biol 371(2):388–395

    Article  CAS  PubMed  Google Scholar 

  11. Crichton PG, Harding M, Ruprecht JJ, Lee Y, Kunji ERS (2013) Lipid, detergent, and Coomassie Blue G-250 affect the migration of small membrane proteins in blue native gels; mitochondrial carriers migrate as monomers not dimers. J Biol Chem 288(30):22163–22173. https://doi.org/10.1074/Jbc.M113.484329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Palmer SM, Kunji ERS (2012) Online monitoring of biomass accumulation in recombinant yeast cultures. Methods Mol Biol 866:165–179. https://doi.org/10.1007/978-1-61779-770-5_14

    Article  CAS  PubMed  Google Scholar 

  13. Palmer SM, Kunji ERS (2012) Online analysis and process control in recombinant protein production (review). Methods Mol Biol 866:129–155. https://doi.org/10.1007/978-1-61779-770-5_12

    Article  CAS  PubMed  Google Scholar 

  14. Thangaratnarajah C, Ruprecht JJ, Kunji ERS (2014) Calcium-induced conformational changes of the regulatory domain of human mitochondrial aspartate/glutamate carriers. Nat Commun 5. https://doi.org/10.1038/ncomms6491

  15. Majd H, King MS, Palmer SM, Smith AC, Elbourne LD, Paulsen IT, Sharples D, Henderson PJ, Kunji ER (2018) Screening of candidate substrates and coupling ions of transporters by thermostability shift assays. elife 7. https://doi.org/10.7554/eLife.38821

  16. Harborne SPD, King MS, Crichton PG, Kunji ERS (2017) Calcium regulation of the human mitochondrial ATP-Mg/Pi carrier SLC25A24 uses a locking pin mechanism. Sci Rep 7:45383. https://doi.org/10.1038/srep45383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hofherr A, Seger C, Fitzpatrick F, Busch T, Michel E, Luan JT, Osterried L, Linden F, Kramer-Zucker A, Wakimoto B, Schutze C, Wiedemann N, Artati A, Adamski J, Walz G, Kunji ERS, Montell C, Watnick T, Kottgen M (2018) The mitochondrial transporter SLC25A25 links ciliary TRPP2 signaling and cellular metabolism. PLoS Biol 16(8). https://doi.org/10.1371/journal.pbio.2005651

  18. Tavoulari S, Thangaratnarajah C, Mavridou V, Harbour ME, Martinou JC, Kunji ER (2019) The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state. EMBO J 38(10). https://doi.org/10.15252/embj.2018100785

  19. Hashimoto M, Shinohara Y, Majima E, Hatanaka T, Yamazaki N, Terada H (1999) Expression of the bovine heart mitochondrial ADP/ATP carrier in yeast mitochondria: significantly enhanced expression by replacement of the N-terminal region of the bovine carrier by the corresponding regions of the yeast carriers. Biochim Biophys Acta Bioenerg 1409(3):113–124. https://doi.org/10.1016/S0005-2728(98)00155-8

    Article  CAS  Google Scholar 

  20. Dvir S, Velten L, Sharon E, Zeevi D, Carey LB, Weinberger A, Segal E (2013) Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast. Proc Natl Acad Sci U S A 110(30):E2792–E2801. https://doi.org/10.1073/pnas.1222534110

    Article  PubMed  PubMed Central  Google Scholar 

  21. Hamilton R, Watanabe CK, de Boer HA (1987) Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs. Nucleic Acids Res 15(8):3581–3593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):31–34. https://doi.org/10.1038/nprot.2007.13

    Article  CAS  PubMed  Google Scholar 

  23. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96

    Article  CAS  PubMed  Google Scholar 

  24. Chae PS, Rasmussen SG, Rana RR, Gotfryd K, Chandra R, Goren MA, Kruse AC, Nurva S, Loland CJ, Pierre Y, Drew D, Popot JL, Picot D, Fox BG, Guan L, Gether U, Byrne B, Kobilka B, Gellman SH (2010) Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7(12):1003–1008. https://doi.org/10.1038/nmeth.1526

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Crichton PG, Lee Y, Ruprecht JJ, Cerson E, Thangaratnarajah C, King MS, Kunji ER (2015) Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers. J Biol Chem 290(13):8206–8217. https://doi.org/10.1074/jbc.M114.616607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Klingenberg M (2009) Cardiolipin and mitochondrial carriers. Biochim Biophys Acta 1788(10):2048–2058

    Article  CAS  PubMed  Google Scholar 

  27. Lee Y, Willers C, Kunji ER, Crichton PG (2015) Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin. Proc Natl Acad Sci U S A 112(22):6973–6978. https://doi.org/10.1073/pnas.1503833112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Horvath SE, Daum G (2013) Lipids of mitochondria. Prog Lipid Res 52(4):590–614. https://doi.org/10.1016/j.plipres.2013.07.002

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

This research was supported by the Medical Research Council (grant MC_UU_00015/1). We thank Drs. Homa Majd and Chancievan Thangaratnarajah for help with preparing figures 1 and 2, respectively.  

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Authors and Affiliations

  1. Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    Martin S. King & Edmund R. S. Kunji

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  1. Martin S. King
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  2. Edmund R. S. Kunji
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Correspondence to Edmund R. S. Kunji .

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Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Camilo Perez

  2. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Timm Maier

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King, M.S., Kunji, E.R.S. (2020). Expression and Purification of Membrane Proteins in Saccharomyces cerevisiae. In: Perez, C., Maier, T. (eds) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0373-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0373-4_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0372-7

  • Online ISBN: 978-1-0716-0373-4

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