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

4. E. coli

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

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. TEV site GFPHisX8GFPHisX8 GAL1 GFPHisX8 TEF Initial tests In two different yeast strains, one with Pep4 deletion In two different promoters, one inducible and the other constitutive

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

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 ConfocalWide field

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

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

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

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

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