GFP-based pipelines for the overexpression and purification of membrane proteins. David Drew
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1 GFP-based pipelines for the overexpression and purification of membrane proteins David Drew
2 1. Produc0on, Purifica0on, Crystalliza0on (Talk 1) GFP- based E. coli pipeline - Bacterial proteins GFP- based S. cerevisiae pipeline - Eukaryo0c proteins Op0miza0on of membrane protein crystals based on detergent stability (Talk 2)
3 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR
4 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR Wagner, S., Lerch- Bader, M., Drew, D., and De Gier J.W., Trends in Biotechnology (2006)
5 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR periplasm cytoplasmic membrane cytoplasm membrane Fluorescent MP- GFP Overexpression inclusion bodies Not Fluorescent Drew, D, Nordlund, P, von Heijne, G, de Gier, JW. FEBS LeU (2001)
6 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR Fluorescent Not Fluorescent periplasm cytoplasmic membrane cytoplasm Drew, D, Sjöstrand, D, Nilsson J, Urbig T, Chin CN, de Gier JW, von Heijne G. (2002). PNAS.
7 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR ~ 80% of E. coli membrane proteins have Cin topology Daley D, Rapp, M, Granseth, E, Melen, K, Drew, D, von Heijne, G (2005) Science Hsieh JM, Besserer GM, Madej MG, Bui HQ, Kwon S, Abramson J. Protein Sci Apr;19(4):
8 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR In- gel fluorescence Drew, D, Lerch, M, Slotboom, D, Kunji, E, de Gier, JW. (2006). Nature Methods
9 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR
10 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR unfolded folded unfolded folded Drew, D, et al. Nature Methods Geertsma ER, et al. PNAS. 2008
11 GFP AS A MEMBRANE PROTEIN FOLDING INDICATOR Green: C41(DE3), Red: C43(DE3), Blue: BL21(DE3)pLysS, odd numbers: 0.1 mm IPTG and even numbers: 0.4 mm IPTG
12 Lemo21(DE3) Wagner et al. (2008) PNAS, 105 (38):
13 Lemo21(DE3)
14 Autoinduction media Gordon et al, (2011). Protein Expr. Purif
15 Eukaryotic Membrane Protein Production Bill, R et al. Nat. Biotechnology (2011)
16 Eukaryotic membrane protein production in S. cerevisiae Newstead S., Hyun, K., von Heijne G., Iwata S., Drew D. (2007). PNAS. Drew, D., Newstead S., Sonoda, Y., Hyun, K., von Heijne G., Iwata S. (2008) Nature Protocols
17 Establishing screen condi4ons Eukaryotic membrane protein production in S. cerevisiae
18 Eukaryotic membrane protein production in S. cerevisiae
19 Eukaryotic membrane protein production in S. cerevisiae yeast cell suspension Add glass beads and break In 0ssue lyser for 10 minutes Spin in table- top centrifuge for 1 hour In- gel fluorescence Run standard SDS- PAGE Suspend crude membranes and measure fluorescence
20 S. cerevisiae overexpression screen strategy Eukaryotic membrane protein production in S. cerevisiae
21 Eukaryotic membrane protein production in S. cerevisiae
22 Screening monodispersity in crude samples by Fluorescence-detection Size-Exclusion Chromatography Kawate, T, Gouaux, E (2006). Structure
23
24
25 ~ 60% of eukaryotic membrane proteins monodisperse in DDM From 15L 13 mg Yeast GDP-mannose tr. 4 mg Human Slc35b1 7 mg Mouse CMP-Sia tr.
26 Detergent solubilization efficiency is a poor criterion for selection 13 / / 17 5 / 17
27 Detergent efficiency NOT good indicator Detergent solubilization efficiency is a poor criterion for selection
28 Detergent solubilization efficiency is a poor criterion for selection
29 Localization
30 ~ 70% targeted to the correct organelle
31 Summary Membrane Proteins produced in S. cerevisiae 25 % of can be overproduced to > 1 mg/l 70 % of those are correctly localized Detergent-solubilization is not a reliable
32 MP- TEV- GFP- His 8 TEV10His 8His Drew, D, Slotboom, D, et al (2005). Prot. Sci.
33
34 Non- solubilized membranes Solubilized membranes Flow- through Ni- NTA bound LacY- GFP LacY- GFP eluate Natural light In- gel fluorescence Coomassie stained
35
36 Example case 1: Rat VGLUT2 Sonoda Y, Cameron A, Newstead S, Omote H, Moriyama Y, Kasahara M, Iwata S, Drew D. FEBS LeU.(2010)
37
38 Example case 2: human GLUT1
39 Crystalliza0on: Test- set of control membrane proteins Sonoda Y et al. Structure. (2011)
40 LDAO AmtB ammonium channel to 1.9-Å 7.0Å 3.5Å 2.3Å 1.8Å
41 9M Mhp1 hydantoin transporter outward facing conformation to 2.8-Å 11.2Å 5.6Å 3.6Å 2.7Å
42 12M NhaA physiological dimer to 3.6-Å 13.6Å 6.5Å 4.3Å 3.2Å
43 12M (BT-9) PepTso -40% identical to PepT at 3.6 / 4.0-Å 14 Å 6.9 Å 4.6 Å 3.4 Å Newstead, S, Drew D., Cameron, A.D., et al. EMBO J. (2011)
44 Benchmarking Stability for Op0mizing Crystalliza0on MP structures from cleavable GFP- His 8-10 tag
45 Transport Systems H + Secondary ac1ve transport Nutrients Symport Na + Nutrients Respira1on H + Na + H + An1port ADP Toxic molecules ATP synthase H + ATP ATP ATP H + Nutrient, K + Peter Henderson Primary ac1ve transport
46 S. Newstead, D. Drew et al. EMBO J. (2011) Secondary Transporters Large fraction of the membrane proteome Play a key role in drug pharmacokinetics Only a few secondary eukaryotic transporter structures known in PDB
47 Transporter structural summary? Crystalliza1on success rate similar between pro- and eukaryo1c transporters. Crystal op1miza1on is the biggest difference. Sonoda Y et al. Structure. (2011)
48 How detergent stable does the MP need to be? Sonoda Y et al. Structure. (2011)
49 Summary Membrane protein production is trial-and-error Fluorescence monitoring facilitates this process Obtaining membrane protein crystals is just the first step
50 Acknowledgements Stockholm University/ CBR Jan- Wllem de Gier (E. coli GFP pipeline) Joy Kim and Gunnar von Heijne (S. cerevisiae GFP pipeline) So Iwata s MPC/MPL group Yo Sonoda, Simon Newstead, Nein- Jen Hu, Alex Cameron, Norimichi Nomura, Hae- Joo Kang My Current group Chiara Lee, Hae- Joo Kang, Nein- Jen Hu
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