Sirandar888

I am currently trying to do soluble expression of a protein in E. coli BL21 and purify it by IMAC.

The yields of soluble protein are not great but good enough but I am having trouble keeping the protein in solution after dialysis.

The protein is a putative metalloprotease and displays some strange behavior and as I am not a protein chemist I don't really know how to interpret it.

The protein is expected to be 38kDa and runs at that on a gel.  Immediately after elution from IMAC and dialysis about 50% of it runs at 38kDa and 50% runs at about 72kDa, probably a dimer.  The interesting part is that neither guanidine nor B-mercapto-ethanol can get the 72kDa band back to 38kDa on a gel.

After a few days the concentration of soluble protein drops from 0.9mg/mL to about 0.4mg/mL and about 80% of the protein runs at ~72kDa.

This behavior seems to be consistent with the protein forming a covalent bond with itself, inactivating itself in the process and precipitating out of solution.

Is this a common occurrence for metalloproteases and what can I do to stop it?  Is there some other explanation for this behavior I have missed?