Extraordinary conclusions require extraordinary proof, yes? If you are completely convinced (!) that auto-proteolysis is possible (meaning that the enzyme you're studying is at least a proteinase in potentia, and also you have utterly ruled out contamination by host proteases) then the next step is to do some rational mutagenesis of putative active site(s) - see if proteolysis stops.
90:10 odds that it's just normal proteolysis - the usual kind, by exogenous proteases - either in the cell before purification, during purification, or during sample handling and crystallization. It happens all the time. Artem - Cosmic Cats approve of this message On Tue, May 26, 2015 at 12:45 AM, Mohammad Khan <mohdkhan0...@gmail.com> 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 >
