Factor Xa Cleavage - MBP-Factor Xa Cleavage (Jul/23/2006 )
Dear all,
Currently I m trying to cleavage the MBP from my fusion protein by using factor xa protease. I purified my protein thru amylose resin, and it has been purified. And then I tried to cleavage the MBP during the purification step (which my fusion protein still bind to the amylose resin), however after I eluted the protein, I lose my protein. Does anyone have any idea to purify and cleavage the MBP fused protein at the same time? Or I have to purify my protein first before proceed to factor xa cleavage? Thank you very much.
Currently I m trying to cleavage the MBP from my fusion protein by using factor xa protease. I purified my protein thru amylose resin, and it has been purified. And then I tried to cleavage the MBP during the purification step (which my fusion protein still bind to the amylose resin), however after I eluted the protein, I lose my protein. Does anyone have any idea to purify and cleavage the MBP fused protein at the same time? Or I have to purify my protein first before proceed to factor xa cleavage? Thank you very much.
What exacgtly d oyu mean by "lose my protein"?
It's easier to purify the fusion protein away from other cellular proteins, then cleave on the column. It means that the major protein component to come out of the column is your protein, relatively pure.
Just to be a bit mischevious, are you sure there aren't any Xa cleavage sites within your protein, and that's why you don't see anything after the digestion step?
Hi there,
got a somewhat similar problem.
expression and purification of MBP-fusion protein works well. Factor Xa digest works nicely either on column or after purification- confirmed by SDS-page gel. The bands of MBP-fusion and MBP only decrease resp. increase over time and are clearly visible. Target protein and one of Factor Xa's subunits run at the same height on SDS gel, therefore difficult to distinguish.
The next step, to purify target protein from digest mix, is problematic. Size exclusion results in one peak, which is Factor Xa and MBP only - (target protein may be there too, but difficult since running at same height as Xa subunit on gel).
It seems like target protein remeins somewhat associated with Xa? Anyone experienced this before? (fusion protein doesn't contain any additional cleavage sites).
Any help would be grately appreciated,
Elke