I assume that the loss of the peptides that you observe for the smaller protein is at the other terminus from the tag? If it is at the terminus where the tag was it would suggest that removal of the tag using proteolysis is the most likely cause. Though saying that Factor Xa/thrombin is not 100% accurate and still maybe causing the slight degradation.
You do not say if you see the two different species before cleavage. If you do, have you tried adding protease cocktail inhibitors before disruption of the cells? As you know which peptides are missing, you know roughly the region that is deleted. Does this cause a change in the pI of the smaller protein? If so you may try ion exchange chromatography to separate the two species. For crystallography purposes, two different species may effect crystallization through disruption of crystal packing, though in situ proteolysis crystallization can generate a mixture of different species with one of the fragments being more readily crystallizable. There is only one way to find out... Daniel A. Bonsor, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 USA
