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

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.