Dear All:
In my case, a 15KD protein without disufide bonds was expressed as
inclusion bodies in E.coli but can be refolded as monomers with a very low
solubility.
Adding glycerol did not help so far.
To increase the solubility, I fused my protein with maltose binding protein.
After fusion, the protein can be solublly expressed but still in an
aggregated form. There is also another problem. A large part of the MBP fusion
protein was degraded somehow even at the condition of low
temperature(18 degree) and low IPTG induction (0.1mM), which resulted in a
truncated form of this protein, 30 out of 140 residues. However, this truncated
form was monomeric.
Based on this observation, I tried several short truncates as well, such as
80, 90, or 120 residues. But, after affinity purification, I got very similar
results as mentioned above.
I checked the sequence of my protein and there is no protease-sensitive
site based on some protease-cutting-site-prediction servers.
So, how can I deal with the degradation problem? and, how can I minimize
the aggregation during expression?
Any suggestions will be highly appreciated.
Have a nice day!
Jerry McCully
By the way, thank folks for the answers of my another question about setting up
view point in Pymol along axis in the unit cell.
