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Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters

  • Louise E. Bird
  • Joanne E. Nettleship
  • Valtteri Järvinen
  • Heather Rada
  • Anil Verma
  • Raymond J. Owens
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 922)

Abstract

The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.

Keywords

Integral membrane protein Green fluorescent protein Insect cells Escherichia coli Saccharomyces cerevisiae Pichia pastoris HEK 293 cells 
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Notes

Acknowledgments

The OPPF-UK is funded by the Medical Research Council, UK (grant MR/K018779/1).

References

  1. Althoff T, Hibbs RE, Banerjee S, Gouaux E (2014) X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors. Nature 512(7514):333–337CrossRefPubMedPubMedCentralGoogle Scholar
  2. Angov E, Hillier CJ, Kincaid RL, Lyon JA (2008) Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host. PLoS ONE 3(5):e2189CrossRefPubMedPubMedCentralGoogle Scholar
  3. Arechaga I, Miroux B, Karrasch S, Huijbregts R, de Kruijff B, Runswick MJ, Walker JE (2000) Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase. FEBS Lett 482(3):215–219CrossRefPubMedGoogle Scholar
  4. Aricescu AR, Owens RJ (2013) Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Curr Opin Struct Biol 23(3):345–356CrossRefPubMedPubMedCentralGoogle Scholar
  5. Baconguis I, Bohlen CJ, Goehring A, Julius D, Gouaux E (2014) X-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a Na(+)-selective channel. Cell 156(4):717–729CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bill RM, Henderson PJF, Iwata S, Kunji ERS, Michel H et al (2011) Overcoming barriers to membrane protein structure determination. Nat Biotechnol 29(4):335–340CrossRefPubMedGoogle Scholar
  7. Bird LE, Rada H, Verma A, Gasper R, Birch J, Jennions M et al (2015) Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli. J Vis Exp 95:e52357Google Scholar
  8. Brillet K, Pereira CA, Wagner R (2010) Expression of membrane proteins in Drosophila Melanogaster S2 cells: production and analysis of a EGFP-fused G protein-coupled receptor as a model. Methods Mol Biol 601:119–133CrossRefPubMedGoogle Scholar
  9. Brooks CL, Morrison M, Joanne Lemieux M (2013) Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris. Protein Sci 22(4):425–433CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE et al (2007) Glycoprotein structural genomics: solving the glycosylation problem. Structure 15(3):267–273CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chaudhary S, Pak JE, Pedersen BP, Bang LJ, Zhang LB et al (2011) Efficient expression screening of human membrane proteins in transiently transfected human embryonic kidney 293S cells. Methods 55(4):273–280CrossRefPubMedPubMedCentralGoogle Scholar
  12. Chaudhary S, Pak JE, Gruswitz F, Sharma V, Stroud RM (2012) Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nat Protoc 7(3):453–466CrossRefPubMedPubMedCentralGoogle Scholar
  13. Chen R (2012) Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 30(5):1102–1107CrossRefPubMedGoogle Scholar
  14. Chen H, Shaffer PL, Huang X, Rose PE (2013) Rapid screening of membrane protein expression in transiently transfected insect cells. Protein Expr Purif 88(1):134–142CrossRefPubMedGoogle Scholar
  15. Darby RA, Cartwright SP, Dilworth MV, Bill RM (2012) Which yeast species shall I choose? Saccharomyces cerevisiae versus Pichia pastoris (review). Methods Mol Biol 866:11–23CrossRefPubMedGoogle Scholar
  16. Drew D, Kim H (2012a) Preparation of Saccharomyces cerevisiae expression plasmids. Methods Mol Biol 866:41–46CrossRefPubMedGoogle Scholar
  17. Drew D, Kim H (2012b) Screening for high-yielding Saccharomyces cerevisiae clones: using a green fluorescent protein fusion strategy in the production of membrane proteins. Methods Mol Biol 866:75–86CrossRefPubMedGoogle Scholar
  18. Drew D, Kim H (2012c) Optimizing Saccharomyces cerevisiae induction regimes. Methods Mol Biol 866:191–195CrossRefPubMedGoogle Scholar
  19. Drew D, von Heijne G, Nordlund P, de Gier JWL (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 507(2):220–224CrossRefPubMedGoogle Scholar
  20. Drew D, Slotboom DJ, Friso G, Reda T, Genevaux P et al (2005) A scalable, GFP-based pipeline for membrane protein overexpression screening and purification. Protein Sci 14(8):2011–2017CrossRefPubMedPubMedCentralGoogle Scholar
  21. Drew D, Lerch M, Kunji E, Slotboom DJ, de Gier JW (2006) Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 3(4):303–313CrossRefPubMedGoogle Scholar
  22. Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, Iwata S (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3(5):784–798CrossRefPubMedPubMedCentralGoogle Scholar
  23. Drews J, Grasmuk H, Unger FM (1973) Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast. Biochem Biophys Res Commun 51(3):804–812CrossRefPubMedGoogle Scholar
  24. Dukkipati A, Park HH, Waghray D, Fischer S, Garcia KC (2008) BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies. Protein Expr Purif 62(2):160–170CrossRefPubMedPubMedCentralGoogle Scholar
  25. Dürr KL, Chen L, Stein RA, De Zorzi R, Folea IM, Walz T, Gouaux E (2014) Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states. Cell 158(4):778–792CrossRefPubMedPubMedCentralGoogle Scholar
  26. Eifler N, Duckely M, Sumanovski LT, Egan TM, Oksche A et al (2007) Functional expression of mammalian receptors and membrane channels in different cells. J Struct Biol 159(2):179–193CrossRefPubMedGoogle Scholar
  27. Frelet-Barrand A, Boutigny S, Kunji ER, Rolland N (2010) Membrane protein expression in Lactococcus lactis. Methods Mol Biol 601:67–85CrossRefPubMedGoogle Scholar
  28. Funes S, Hasona A, Bauerschmitt H, Grubbauer C, Kauff F et al (2009) Independent gene duplications of the YidC/Oxa/Alb3 family enabled a specialized cotranslational function. Proc Natl Acad Sci U S A 106(16):6656–6661CrossRefPubMedPubMedCentralGoogle Scholar
  29. Funes S, Kauff F, van der Sluis EO, Ott M, Herrmann JM (2011) Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts. Biol Chem 392(1–2):13–19PubMedGoogle Scholar
  30. Giacalone MJ, Gentile AM, Lovitt BT, Berkley NL, Gunderson CW, Surber MW (2006) Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. Biotechniques 40(3):355–364CrossRefPubMedGoogle Scholar
  31. Goehring A, Lee CH, Wang KH, Michel JC, Claxton DP et al (2014) Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat Protoc 9(11):2574–2585CrossRefPubMedPubMedCentralGoogle Scholar
  32. Gordon E, Horsefield R, Swarts HG, de Pont JJH, Neutze R, Snijder A (2008) Effective high-throughput overproduction of membrane proteins in Escherichia coli. Protein Expr Purif 62(1):1–8CrossRefPubMedGoogle Scholar
  33. Gul N, Linares DM, Ho FY, Poolman B (2014) Evolved Escherichia coli strains for amplified, functional expression of membrane proteins. J Mol Biol 426(1):136–149CrossRefPubMedGoogle Scholar
  34. Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y et al (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313(5792):1441–1443CrossRefPubMedGoogle Scholar
  35. He Y, Wang K, Yan N (2014) The recombinant expression systems for structure determination of eukaryotic membrane proteins. Protein Cell 5(9):658–672CrossRefPubMedPubMedCentralGoogle Scholar
  36. Hu NJ, Rada H, Rahman N, Nettleship JE, Bird L et al (2015) GFP-based expression screening of membrane proteins in insect cells using the baculovirus system. Methods Mol Biol 1261:197–209CrossRefPubMedGoogle Scholar
  37. Iost I, Guillerez J, Dreyfus M (1992) Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo. J Bacteriol 174(2):619–622CrossRefPubMedPubMedCentralGoogle Scholar
  38. Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14(4):673–681CrossRefPubMedGoogle Scholar
  39. Kawate T, Michel JC, Birdsong WT, Gouaux E (2009) Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. Nature 460(7255):592–598CrossRefPubMedPubMedCentralGoogle Scholar
  40. King MS, Boes C, Kunji ER (2015) Membrane protein expression in Lactococcus lactis. Methods Enzymol 556:77–97CrossRefPubMedGoogle Scholar
  41. Klepsch MM, Persson JO, De Gier JWL (2011) Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli. J Mol Biol 407(4):532–542CrossRefPubMedPubMedCentralGoogle Scholar
  42. Kunji ER, Slotboom DJ, Poolman B (2003) Lactococcus lactis as host for overproduction of functional membrane proteins. Biochim Biophys Acta 1610(1):97–108CrossRefPubMedGoogle Scholar
  43. Lagane B, Gaibelet G, Meilhoc E, Masson JM, Cézanne L, Lopez A (2000) Role of sterols in modulating the human mu-opioid receptor function in Saccharomyces cerevisiae. J Biol Chem 275(43):33197–33200CrossRefPubMedGoogle Scholar
  44. Lee JK, Stroud RM (2010) Unlocking the eukaryotic membrane protein structural proteome. Curr Opin Struct Biol 20(4):464–470CrossRefPubMedPubMedCentralGoogle Scholar
  45. Lee C, Kang HJ, Hjelm A, Qureshi AA, Nji E, Choudhury H et al (2014a) MemStar: a one-shot Escherichia coli-based approach for high-level bacterial membrane protein production. FEBS Lett 588(20):3761–3769CrossRefPubMedGoogle Scholar
  46. Lee C, Yashiro S, Dotson DL, Uzdavinys P, Iwata S et al (2014b) Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights. J Gen Physiol 144(6):529–544CrossRefPubMedPubMedCentralGoogle Scholar
  47. Lee CH, Lü W, Michel JC, Goehring A, Du J, Song X, Gouaux E (2014c) NMDA receptor structures reveal subunit arrangement and pore architecture. Nature 511(7508):191–197CrossRefPubMedPubMedCentralGoogle Scholar
  48. Linares DM, Geertsma ER, Poolman B (2010) Evolved Lactococcus lactis strains for enhanced expression of recombinant membrane proteins. J Mol Biol 401(1):45–55CrossRefPubMedGoogle Scholar
  49. Logez C, Alkhalfioui F, Byrne B, Wagner R (2012) Preparation of Pichia pastoris expression plasmids. Methods Mol Biol 866:25–40CrossRefPubMedGoogle Scholar
  50. Loll PJ (2003) Membrane protein structural biology: the high throughput challenge. J Struct Biol 142(1):144–153CrossRefPubMedGoogle Scholar
  51. Marreddy RK, Geertsma ER, Permentier HP, Pinto JP, Kok J, Poolman B (2010) Amino acid accumulation limits the overexpression of proteins in Lactococcus lactis. PLoS ONE 5(4):e10317CrossRefPubMedPubMedCentralGoogle Scholar
  52. Milic D, Veprintsev DB (2015) Large-scale production and protein engineering of G protein-coupled receptors for structural studies. Front Pharmacol 6:66PubMedPubMedCentralGoogle Scholar
  53. Miroux B, Walker JE (1996) 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 260(3):289–298CrossRefPubMedGoogle Scholar
  54. Molbaek K, Scharff-Poulsen P, Helix-Nielsen C, Klaerke DA, Pedersen PA (2015) High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae. Microb Cell Fact 14:15CrossRefPubMedPubMedCentralGoogle Scholar
  55. Mus-Veteau I (2010) Heterologous expression of membrane proteins for structural analysis. Methods Mol Biol 601:1–16CrossRefPubMedGoogle Scholar
  56. Newstead S, Kim H, von Heijne G, Iwata S, Drew D (2007) High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 104(35):13936–13941CrossRefPubMedPubMedCentralGoogle Scholar
  57. Nji E, Li D, Doyle DA, Caffrey M (2014) Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease from Pseudomonas aeruginosa. Acta Crystallogr F Struct Biol Commun 70(10):1362–1367CrossRefPubMedPubMedCentralGoogle Scholar
  58. Parcej D, Guntrum R, Schmidt S, Hinz A, Tampé R (2013) Multicolour fluorescence-detection size-exclusion chromatography for structural genomics of membrane multiprotein complexes. PLoS ONE 8(6):e67112CrossRefPubMedPubMedCentralGoogle Scholar
  59. Pedersen PA, Rasmussen JH, Jørgensen PL (1996) Expression in high yield of pig alpha 1 beta 1 Na, K-ATPase and inactive mutants D369N and D807N in Saccharomyces cerevisiae. J Biol Chem 271(5):2514–2522CrossRefPubMedGoogle Scholar
  60. Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8(6):423–488CrossRefPubMedGoogle Scholar
  61. Scharff-Poulsen P, Pedersen PA (2013) Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography. PLoS ONE 8(10):e76851CrossRefPubMedPubMedCentralGoogle Scholar
  62. Schlegel S, Löfblom J, Lee C, Hjelm A, Klepsch M et al (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J Mol Biol 423(4):648–659CrossRefPubMedGoogle Scholar
  63. Schlegel S, Hjelm A, Baumgarten T, Vikström D, de Gier JW (2014) Bacterial-based membrane protein production. Biochim Biophys Acta 1843(8):1739–1749CrossRefPubMedGoogle Scholar
  64. Sonoda Y, Newstead S, Hu NJ, Alguel Y, Nji E et al (2011) Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19(1):17–25CrossRefPubMedPubMedCentralGoogle Scholar
  65. Thomas J, Tate CG (2014) Quality control in eukaryotic membrane protein overproduction. J Mol Biol 426(24):4139–4154CrossRefPubMedPubMedCentralGoogle Scholar
  66. Wagner S, Bader ML, Drew D, de Gier JW (2006) Rationalizing membrane protein overexpression. Trends Biotechnol 24(8):364–371CrossRefPubMedGoogle Scholar
  67. Wagner S, Baars L, Ytterberg AJ, Klussmeier A, Wagner CS et al (2007) Consequences of membrane protein overexpression in Escherichia coli. Mol Cell Proteomics 6(9):1527–1550CrossRefPubMedGoogle Scholar
  68. Wagner S, Bader ML, Drew D, de Gier JW (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105(38):14371–14376CrossRefPubMedPubMedCentralGoogle Scholar
  69. Wang KH, Penmatsa A, Gouaux E (2015) Neurotransmitter and psychostimulant recognition by the dopamine transporter. Nature 521(7552):322–327CrossRefPubMedPubMedCentralGoogle Scholar
  70. Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E (2005) Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437(7056):215–223CrossRefPubMedGoogle Scholar
  71. Yang H, Murphy AS (2009) Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe. Plant J 59(1):179–191CrossRefPubMedGoogle Scholar
  72. Yang Y, Hu Z, Liu Z, Wang Y, Chen X, Chen G (2009) High human GLUT1, GLUT2, and GLUT3 expression in Schizosaccharomyces pombe. Biochemistry (Mosc) 74(1):75–80CrossRefGoogle Scholar
  73. Zhang F, Manzan MA, Peplinski HM, Thiem SM et al (2008) A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system. Vitro Cell Dev Biol Anim 44(7):214–223CrossRefGoogle Scholar
  74. Zweers JC, Barák I, Becher D, Driessen AJ, Hecker M et al (2008) Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes. Microb Cell Fact 7:10CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Louise E. Bird
    • 1
    • 2
  • Joanne E. Nettleship
    • 1
    • 2
  • Valtteri Järvinen
    • 1
    • 2
  • Heather Rada
    • 1
    • 2
  • Anil Verma
    • 1
    • 2
  • Raymond J. Owens
    • 1
    • 2
  1. 1.OPPF-UK, The Research Complex at Harwell, Rutherford Appleton Laboratory HarwellOxfordUK
  2. 2.Division of Structural Biology, Henry Wellcome Building for Genomic MedicineUniversity of OxfordOxfordUK

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