On Sep 8, 2010, at 6:31 AM, Jerry McCully wrote: > 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.
Hi Jerry, I'm afraid that, at least in our hands, the behavior that you describe generally means that a given passenger protein is largely unfolded - although it still manages to stay in solution thanks to the well-known solubilization power of MBP. This, of course, usually also leads to aggregation - exactly like you are seeing. Because of this, subjecting rigid MBP fusions to limited proteolysis is one of the first things we do, when we are characterizing new constructs and want to make sure that the passenger is properly folded. Luca ---------------------------------------------------------------- Luca Jovine, Ph.D. Group Leader & EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition & Center for Biosciences Hälsovägen 7, SE-141 57 Huddinge, Sweden Voice: +46.(0)8.6083-301 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org ----------------------------------------------------------------
