High-throughput, flourescent-based optimisation of eukaryotic membrane protein overexpression

and purification.

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Simon Newstead, Joy Kim, Gunnar von Heijne, So Iwata, David Drew

Eukaryotic membrane proteins

• Eukaryotic membrane proteins are difficult to produce in large quantities > 1mg.

• Important to obtain structural information.• Currently only three structures in the PDB from heterologous overexpression– Potassium Channel, Spinach Aquaporin, Ca2+ ATPase.

• We needed to develop a cost effective, high-throughput approach to screening in our group.

• Developed a GFP-based fusion system, in combination with SEC to screen 43 eukaryotic MPs.

Questions that needed addressing:

1. Can the target protein be expressed to high levels > 1mg/ml?

2. Is the protein stable?

3. Can we optimise the expression?

4. What is the quality of the protein?

5. Which detergents should be used to extract and set up crystal screens?

6. Shortest time possible!

MP-GFP overexpression

N

C

GFP

inclusion bodiesmembrane

N

CGFP

N

CGFP

N

CGFP

cytoplasmicmembrane

cytoplasm

periplasm

Drew, D, Nordlund, P, von Heijne, G, de Gier, JW. FEBS Lett (2001)

GFP: Proven membrane protein folding indicator in E. coli

Daley DO, Rapp, M, Granseth, E, Melen, K, Drew, D, von Heijne, G (2005) Science

GFP: Used to monitor expression of E. coli inner membrane proteome

Drew, D, Lerch, M, Slotboom, D, Kunji, E, de Gier, JW. (2006). Nature Methods

GFP: Is stable in standard SDS-PAGE and replaces western blotting

0

0.5

1

1.5

2

2.5

3

Fur4 Azr1AGP1Isc1

Hsp30Sec61YOL162W

ITR1Shr3pHNM1HXT3HXT1UGA4MPH3ARN1DAL4TPO4DUR3KHA1Rft1SsH1 GAL

S. cerevisiae overexpression screen strategyQ1: Does the target protein express well?

Best strain vs promoter combination established by testing expression of 88 yeast transport proteins

Expression estimate in yeast whole-cells correlates well with estimate in membranes

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Yeast cell suspension Add glass beads and breakIn tissue lyser for 10 minutes

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Spin in table-topcentrifuge for 1 hour

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Suspend crude membranesand measure fluorescence

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Run standard SDS-PAGE

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In-gel fluorescence

S. cerevisiae overexpression screen strategyQ2: Is the target protein stable?

In-gel fluorescence of crude membranes to verifystability

In gel flourescence correlates very well to fluorescence in the membranes

S. cerevisiae overexpression screen strategy Q3: Can we improve the overexpression further?

Good correlation when scaled up to fermenters.

S. cerevisiae overexpression screen strategy Q4: How is the quality of the protein under expression conditions?

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Kawate, T, Gouaux, E (2006). Structure

S. cerevisiae overexpression screen strategyQ5: How is quality of the protein with different detergents ?

Use less than 1% of sample to screen detergent

GFP-based detergent screen to measure extraction efficiency

Some examples:

Summary.

• Based on the expression of 43 Eukaryotic membrane proteins we present a cost effective high throughput approach to screening.

• We find that 70 % of the well expressed MPs tested in this system are stable, targeted to the correct organelle and monodisperse in either FC-12 or DDM.

• We hope to get some of our targets into crystals suitable for X-ray diffraction analysis.

• Soluble proteins could be screened just as effectively.