On Mon, 17 Jul 1995, Flip Hoedemaeker wrote:
> Hi, I have a fusion protein with maltose-binding protein and I seem to have
> trouble in cutting off the MBP part with factor Xa. I have heared that other
> people have this problem, and that it can be due to poor accesibility of the
> cleavage site. One way of improving cleavage would be to (partially) unfold the
> fusion protein, e.g. with urea. Of course, factor Xa would still have to be
> active.
> Does anyone out there have experience with this kind of experiment, and do you
> know at what urea concentrations Xa is still active? Any other ideas?
> Thanks, Flip
>>> I have not used Factor Xa in the presence of urea, but have
successfully used it to cleave fusion proteins in the presence of low
concentrations (0.02%) of SDS. In my case, I first denatured the fusion
protein with 2% SDS and then diluted it to 0.02% before addition of FXa.
However, I've read somewhere that even the low SDS concentration may help
with cleavage without prior denaturation at higher concentrations. I hope
this helps.
Joe
