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GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae

GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae. Joy Kim. Center for Biomembrane Research Department of Biochemistry and Biophysics Stockholm University Stockholm, Sweden. Soluble protein vs. membrane protein structures. GFP-based.

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GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae

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  1. GFP-based membrane protein overexpression and purification in E. coli and S. cerevisiae Joy Kim Center for Biomembrane Research Department of Biochemistry and Biophysics Stockholm University Stockholm, Sweden

  2. Soluble protein vs. membrane protein structures

  3. GFP-based Overexpression screening Overexpression optimization Detergent screening Homologs of a target protein Solubilization with suitable detergent Purification Crystallization Structure determination

  4. E. coli

  5. WB In-gel fluorescence vs. Western Blotting IGF

  6. Overexpression of eukaryotic membrane proteins in E. coli • Differences in lipid compositions • Post translational modifications • Folding environments • Alternative overexpression hosts • Yeast (P. pastoris, S. cerevisiae) • Insect and mammalian cells • Cell-free system • Easy genetic manipulations • Well known genetics • ER quality control system

  7. GFP GFP GFP HisX8 HisX8 HisX8 GAL1 TEF Initial tests TEV site In two different promoters, one inducible and the other constitutive In two different yeast strains, one with Pep4 deletion

  8. Fluorescence of whole-cell lysates from 10 ml vs. 1L cultures

  9. Comparison of the upscale expression in 2.5 L shaker flask vs. 15 L fermenter

  10. Time course for the overexpression of MP-GFP fusions

  11. Fluorescence of whole-cell lysates vs. membrane fractions

  12. Fluorescence in solution vs. in-gel fluorescence Fluorescence In gel band intensity

  13. Combination of Gal1, an inducible promoter/Pep4, a vacuolar protease deletion strain gave the overall best overexpression for majority of test membrane proteins. Overexpression screening of yeast MP-GFP fusions

  14. Optimizing expression with chemical chaperones

  15. N-terminal vs. C-terminal GFP tagging 1: Rer1-GFP 2: GFP-Rer1

  16. Assessing the quality of MP-GFP fusions by subcellular localization Confocal Wide field

  17. Assessing the quality of MP-GFP fusions by FSEC MP-GFP fusion Free GFP Aggregation Newstead et al., 2007, PNAS 104:13936-41

  18. Purification of a nucleotide sugar transporter Detergent solubilized membranes Purified MP-GFP Purified MP Newstead et al., 2007, PNAS 104:13936-41

  19. GFP-based overexpression and purification of eukaryotic membrane proteins in S. cerevisiae Drew et al., Nat. Protocol, in press

  20. GFP-based Homologs of a target protein Overexpression screening Overexpression optimization Solubilization with suitable detergent Detergent screening Purification Crystallization Structure determination

  21. Acknowledgements Center for Biomembrane Research Stockholm University Stockholm, Sweden Membrane Crystallography group Imperial College London, U. K. David Drew Simon Newstead So Iwata Jan Willem de Gier Gunnar von Heijne

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