To dear Herman, Thank you very much for your comments, I do include protease inhibitor in the protein sample (I use Roche, which always works fine for me), and that's why I am so surprised to see that it degrades madly. On the other hand, since the protein behaves quite well on SEC --- nice symmetrical peak, resonable oligomeric state (dimer), I never thought of misfolding problem, but you're right, I should check the proteins' secondary structure first. Thanks again.
To dear Chris, Thank you very much for your advise of adding GSSH, and thank you so much for telling me a working concentration. Best, Bei 2011/8/19 Chris Meier <[email protected]> > Bei: > > My experience confirms Herman's comment -- your protein may be unfolded or > misfolded. > > In addition to protease inhibitors, you could also add an oxidising agent > (e.g. 10mM GSSH) to the protein buffer. > In my experience this keeps many disulphide-bonded proteins happy and > stable. > > Hope this helps. > > Best wishes, > Chris > > ---------------------------------------------------------------- > > From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of > [email protected] > Sent: Fri, August 19,2011 8:55 > To: [email protected] > Subject: Re: [ccp4bb] Off topic_protein degradation. > > Dear Bei, > > The first question to ask is not whether there is spontaneous disulfide > bond breakage, but whether the correct disulfide bonds have been made in the > first place. Extreme protease sensitivity could point to an > unfolded/misfolded protein. If you know some protein NMR people, you could > ask them to check. Even a 1-D NMR spectrum could give some information > whether the protein is correctly folded or not. Another way to check is to > see if your protein has proper enzymatic/biological activity. If this > activity is ok, the folding is probably ok as well. > > You may have a protease contaminant, so you may want to check the > purification protocol. The least you could do is to add some protease > inhibitors to your crystallization setups. I once added PMSF for this > purpose. > > Good luck! > Herman > > From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of > joybeiyang > Sent: Friday, August 19, 2011 6:28 AM > To: [email protected] > Subject: [ccp4bb] Off topic_protein degradation. > Dear all, > > I am trying to crystallize a protein for which the yield and solubility > were both fine. However, this protein has a severe problem of degradation. > When stored at RT, the protein will degrade madly into pieces, while stored > at 4 degree, the degradation is much slower and a relatively stable truncate > form can be get. I am going to try to crystallize the protein at 4 degree, > however I still want to understand what's going on there at RT because this > protein was supposed to be very stable, it is Cys rich, and the 6 Cys were > predicted to form 3 disulfide bonds which hold the protein as a globule, how > can a protein with 3 stabilizing disulfide bond be fully degraded like this? > Is there a possibility of spontaneous disulfide bond breakage at pH 8 ? > > Another question is I tried limited proteolysis with this protein, however > even at 1:1000(w/w, chymotrypsin), the protein is degraded into pieces in > about 2 hrs (again, a relatively stable truncated form can be get between 10 > min and 30 min). I am wondering how is the crystallization probability > correlated with proteolysis stability? Does this phenomenon indicate that > the crystallization probability of my protein is pretty low? > > Any comments would be greatly appreciated. > > Bei > > 2011-08-15 >
