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Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden Green Light for Membrane Proteins.

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Presentation on theme: "Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden Green Light for Membrane Proteins."— Presentation transcript:

1 Jan-Willem de Gier Center for Biomembrane Research, Stockholm, Sweden Green Light for Membrane Proteins

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

3 Monitoring overexpression of a YedZ-GFP fusion using in-gel and whole-cell fluorescence David Drew et al. Nat Methods (2006) 4:303-13

4 From detergent screen to recovered membrane protein David Drew et al. Nat Methods (2006) 4:303-13

5 Recovered membrane proteins can be used for functional and structural studies David Drew et al. Protein Sci (2005) 14: 2011-2017

6 Simon Newstead et al. PNAS (2007) 104(35):13936-41 GFP-based pipeline in yeast

7 Biogenesis of membrane proteins in E. coli Samuel Wagner et al. (2006) Trends Biotechnol 24: 364-371 membrane proteins secretory proteins

8 E. coli BL21(DE3)/pET overexpression system Merck Novagen, pET manual

9 Experimental setup (1) Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

10 Experimental setup (2) Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

11 Consequences of membrane protein overexpression in E. coli (1) Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

12 Consequences of membrane protein overexpression in E. coli (2) Samuel Wagner et al. (2007) Mol Cell Proteomics (9) 1527-50.

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

14 Differences in overexpression kinetics? Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

15 Differences in overexpression kinetics

16 Effect of the addition of IPTG Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

17 Mutations in the promoter governing T7RNAP expression in C41(DE3) and C43(DE3) Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

18 Mimicking the effect of the mutations in the promoters with T7 lysozyme Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

19 Mimicking the effect of the mutations in the promoters with T7 lysozyme Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

20 Characterization of Lemo21(DE3) Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

21 Lemo21(DE3) is an ’all in one’ platform for membrane protein overexpression Samuel Wagner et al. (2008) PNAS, 105 (38):14371-6.

22

23 Funding -Swedish Research Council (VR) -Wallenberg Foundation -Swedish Foundation for Strategic Research (SSF) -National Institutes of Health (NIH) www.xbranebio.com www.cbr.su.se


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