I've tried 4 strains: Arctic Express, Rosetta, Origami, and BL21. None of them could make my enzymes soluble.
I did enzymatic experiments on the pellet lysate (where the inclusion bodies are present) and the enzymes is working. So, has anyone an idea how to recover the enzymes from it? A protocol would be very helpful.
Thanks
Did you tried low temperature (18 or 16 degree) induction overnight. Generally if you purifying eukaryotic enzyme, BL21 codon plus is the preferred choice along with Rosetta. You can follow the qiagen protocols for purifying from inclusion body.
Perhaps the same protocol whereby you got soluble enzyme activity from the pellets? Making the protein soluble in the cells often requires low expression and cold temperatures. Any S-S bonds in the enzyme?
It's possible that the protein is in the form of aggregates and is therefore in the pellet, but is not in the inclusion bodies. When diluted into the enzyme assay buffer, enough of it may dissociate from the aggregates to show some enzyme activity. It may be worth trying to extract the pellet with buffer. Choosing the right pH, salt concentration, and non-ionic detergent could help.
If the protein is mostly in inclusion bodies, you could try dissolving them in urea or guanidine hydrochloride, then refolding the protein.
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