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?
Sirandar888Member Since 18 Jan 2013
Offline Last Active Jan 22 2013 02:48 PM
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Protein Purification, Pathobiology
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22 Apr 2013 - 03:17