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
Solubilization with suitable detergent
Purification
Crystallization
Structure determination
Homologs of a target protein Overexpression screening
Overexpression optimization
Detergent screening
GFP-based
E. coli
In-gel fluorescence vs. Western Blotting
IGF WB
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
TEV site
GFP HisX8
GFP HisX8GAL1
GFP HisX8TEF
Initial tests
In two different yeast strains, one with Pep4 deletion
In two different promoters, one inducible and the other constitutive
Fluorescence of whole-cell lysates from 10 ml vs. 1L cultures
Comparison of the upscale expression in 2.5 L shaker flask vs. 15 L fermenter
Time course for the overexpression of MP-GFP fusions
Fluorescence of whole-cell lysates vs. membrane fractions
Fluorescence
In gel band intensity
Fluorescence in solution vs. in-gel fluorescence
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
Optimizing expression with chemical chaperones
N-terminal vs. C-terminal GFP tagging
1: Rer1-GFP2: GFP-Rer1
Assessing the quality of MP-GFP fusions by subcellular localization
Confocal Wide field
Assessing the quality of MP-GFP fusions by FSEC
Newstead et al., 2007, PNAS 104:13936-41
Aggregation
MP-GFP fusion
Free GFP
Purification of a nucleotide sugar transporter
Newstead et al., 2007, PNAS 104:13936-41
Detergent solubilized membranes Purified MP-GFP Purified MP
GFP-based overexpression and purification of eukaryotic membrane proteins in S. cerevisiae
Drew et al., Nat. Protocol, in press
Overexpression screening
Solubilization with suitable detergent
Purification
Crystallization
Structure determination
Overexpression optimization
Detergent screening
Homologs of a target protein
GFP-based
Acknowledgements
Jan Willem de GierGunnar von Heijne
David DrewSimon NewsteadSo Iwata
Center for Biomembrane ResearchStockholm UniversityStockholm, Sweden
Membrane Crystallography groupImperial College
London, U. K.