Purifiying insoluble protein

[ANDY PHILLIPS]

We're trying to express a cytochrome P450 in E.coli to investigate its 
activity. We've more or less given up hope that we will get assembled, active 
enzyme, but we can get lots of insoluble protein, presumably in inclusion 
bodies. We'd like to purify this to raise antibodies, so that we can try to 
precipitate activity from plant extracts. However, we're having great 
difficulty in dissolving the protein for purification. We've tried buffers, 
detergents, 8M urea, but no go. The only things that work are SDS and sarkosyl. 
When we dialyze the solubilized protein it precipitates. We've tried 
ion-exchange on the sarkosyl-dissolved material, but it won't come off until 
1M NaCl, with no purification.

Any ideas? We're about to give up and use prep SDS gels, as the recombinant 
band is pretty well separated.

Andy

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