Hi, We want to produce a protein with a SUMO tag, that is known that needs Gly-Gly at the C terminal of the protease site to cleave especifically and release the protein target without any remaining aa. Our protein needs a Gly at the N-terminus. So, do anybody knows what will happen if we add an aditional Gly after the Gly-Gly of the protease site? Will I obtain a protein with a Gly in the N-term after cleavage? or the protease will cleave the third Gly?
Hope that is enough clear.....
you will get the cleaved protein with one Gly at the N-term, the same as with any other amino acid after the terminal 2 Gly of SUMO (except Pro which is not cleaved). We haven't done MS with all of our SUMO-constructs after cleavage but for those which we have checked by MS, we didn't find any indication of cleaving AFTER the first N-terminal amino acid of the target protein.
In one case, there was a minor peak that would correspond to an additional Gly on the target protein, so maybe there is a small chance of cleavage between the 2 terminal Glycins. We haven't checked this in more detail I must admit.
Thanks a lot!!