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Jan-Willem de Gier

Center for Biomembrane Research, Stockholm, Sweden

Green Light for Membrane Proteins

MP-GFP overexpression

N

C

GFP

inclusion bodiesmembrane

N

CGFP

N

CGFP

N

CGFP

cytoplasmicmembrane

cytoplasm

periplasm

David Drew et al. Nat Methods (2006) 4:303-13

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Monitoring overexpression of a YedZ-GFP fusion using in-gel and whole-cell fluorescence

David Drew et al. Nat Methods (2006) 4:303-13

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From detergent screen to recovered membrane protein

David Drew et al. Nat Methods (2006) 4:303-13

C

N

MP-TEV-GFP-8His

GFP

TEV10His

8His

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Recovered membrane proteins can be usedfor functional and structural studies

David Drew et al. Protein Sci (2005) 14: 2011-2017

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0

0.5

1

1.5

2

2.5

3

Fur4 Azr1AGP1Isc1

Hsp30Sec61YOL162W

ITR1Shr3pHNM1HXT3HXT1UGA4MPH3ARN1DAL4TPO4DUR3KHA1Rft1SsH1 GAL

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Simon Newstead et al. PNAS (2007) 104(35):13936-41

GFP-based pipeline in yeast

Biogenesis of membrane proteins in E. coli

Samuel Wagner et al. (2006) Trends Biotechnol 24: 364-371

membrane proteins secretory proteins

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E. coli BL21(DE3)/pET overexpression system

Merck Novagen, pET manual

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Experimental setup (1)

Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

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Experimental setup (2)

Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

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Consequences of membrane protein overexpression in E. coli (1)

Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

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Consequences of membrane protein overexpression in E. coli (2)

Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

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Characterization of C41(DE3) and C43(DE3), also known as the Walker strains

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Differences in overexpression kinetics?

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Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

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Differences in overexpression kinetics

Effect of the addition of IPTG

Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

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Mutations in the promoter governing T7RNAP expression in C41(DE3) and C43(DE3)

Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

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Mimicking the effect of the mutations in the promoters with T7 lysozyme

Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

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Mimicking the effect of the mutations in the promoters with T7 lysozyme

Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

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Characterization of Lemo21(DE3)

Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

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Lemo21(DE3) is an ’all in one’ platform for membrane protein overexpression

Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

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Funding-Swedish Research Council (VR)-Wallenberg Foundation-Swedish Foundation for Strategic Research (SSF)-National Institutes of Health (NIH)

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www.xbranebio.com

www.cbr.su.se

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