Hi Mohammad, I'm not sure anyone here can give you absolute answers, because we don't know enough about your system, but there's 3 things you might want to explore:
1. Contaminating proteases, even at tiny amounts, could lead to appreciable cleavage of your protein over the length of a crystallization trial. Trying to identify a protease and/or cleavage site is not easy, but if absolutely necessary you can maybe try Edman sequencing the domain to try to identify the cut site. 2. In some cases, nonspecific chemical cleavage can happen at aspartate residues, especially if you are at Asp-Pro, and if it is in a disordered region, at acidic pH. http://www.sciencedirect.com/science/article/pii/0076687977470174 3. Like you mention, your protein could be cutting itself. This would be the most interesting case, especially because it might imply some biological function. That said, you'd have to design some experiments to test if this were the case, before coming to that conclusion over the more likely case of protease contamination. All that said, does it matter why your protein is being cleaved? If you have crystals containing your full-length protein, could that be enough? Think about why you need to know the source of this cleavage, and that might point you in the right direction. Mass spec and Edman sequencing might give you hints about whether the cleavage is specific or not, and further purification would remove contaminating proteases, if that is the cause of your cleavage. There's also a possibility that your protein is being cleaved in the cell before you purify - in that case, changing the growth conditions might reduce that cleavage, and further purification could remove fragments. Running a time course digest on a gel could indicate whether the proteolysis is happening before or after purification. Hope this helps, Shane Caldwell McGill University On Tue, May 26, 2015 at 1:45 AM, Mohammad Khan <[email protected]> wrote: > Dear all, > > I am working on a His-tag recombinant protein with two domains, which I > purify using affinity chromatography. When I set up crystallization of the > same, it gave me crystals in two different conditions- one was the complete > protein. The other just had the Domain2. > > Even on SDS-PAGE, I see three different bands of the purified protein. > > How can I determine the autu-proteolysis of this protein? This class of > protein has not reported to have any auto-proteolytic properties, though it > forms isopeptide bonds by autocatalysis. > > Thanks a lot > > Mohammad >
