High-throughput, flourescent-based optimisation of eukaryotic membrane protein overexpression and purification. 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, Ca 2+ 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!

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

S. cerevisiae overexpression screen strategy Q1: 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

Yeast cell suspensionAdd glass beads and break In tissue lyser for 10 minutes Spin in table-top centrifuge for 1 hour Suspend crude membranes and measure fluorescence Run standard SDS-PAGEIn-gel fluorescence S. cerevisiae overexpression screen strategy Q2: Is the target protein stable?

In-gel fluorescence of crude membranes to verify stability

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?

Kawate, T, Gouaux, E (2006). Structure S. cerevisiae overexpression screen strategy Q5: 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.