Bacterially expressed protein in inclusion bodies- you may be screwed!

First try lower temperature (e.g. 22°C) or lower IPTG (e.g. 0.1mM)

Then try

Solubilisation by alkaline extraction (courtesy of Ian Prior).

Keep on ice throughout.

For a pellet of lysed cells obtained from a 1 litre culture.

1. Resuspend pellet in 10ml (at least 5 vols) of 10mM EDTA, pH 8.0;

briefly sonicate with a probe sonicator.

2. pellet 20,000g, 15 min, 4°C

3. repeat 2 more times

4. resuspend the pellet in 2.5ml of 10mM

EDTA, pH 8.0, 1mM DTT; briefly sonicate (the suspension should be milky white).

5. Add an equal volume (2.5ml) of 50mM NaOH, sonicate briefly (the

suspension will have partially cleared; don't leave for long at high

pH).

6. Rapidly neutralise by adding 4 volumes (20ml) of 25mM Tricine, pH

8.0, 10% glycerol, 1mM EDTA, 1mM DTT, + protease inhibitors.

7. Leave on ice for 1 hour. Some proteins will reprecipitate.

8. Pellet out insoluble, 20,000g, 30 mins, 4°C.

If this was GFP, the supernatant would now be bright green and contain 50-70mg of GST-GFP in 25-30ml with relatively little contamination of other proteins.

If this doesn’t work proceed to Sf9 cell expression or work on another protein.