factor Xa and urea?

Joseph Boothe jbooth at ACS.UCALGARY.CA
Thu Jul 20 16:05:55 EST 1995



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



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