Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden
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Transcript of Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden
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Jan-Willem de Gier
Center for Biomembrane Research, Stockholm, Sweden
Green Light for Membrane Proteins
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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
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1
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Fur4 Azr1AGP1Isc1
Hsp30Sec61YOL162W
ITR1Shr3pHNM1HXT3HXT1UGA4MPH3ARN1DAL4TPO4DUR3KHA1Rft1SsH1 GAL
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Simon Newstead et al. PNAS (2007) 104(35):13936-41
GFP-based pipeline in yeast
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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.
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Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.
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Differences in overexpression kinetics
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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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