Hello Every one,
I am trying to purify a human protein in a
bacterial expression system of around 82 kDa (with a 5 kDa His tag, so
fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem
is I am not able to get rid of the infamous contamination proteins of arnA
gene (72 kDa) and glmS gene (67 kDa).
I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa).
This TEV protease has N- terminal His tag. So I first elute my fusion
protein with higher imidazole concentration and then do TEV cleavage by
adding TEV protease,, but sadly It co-elutes other contamination proteins
such as 72 kDa and 67 kDa, as mentioned above..
Now, I wanted to know,,, can I do "On beads cleavage" by directly adding
TEV enzyme when the fusion protein is still bound to Ni-NTA beads??
But I am worreid TEV protease which has N- terminal His tag also try to
bind on Ni-NTA beads..
Please help me..
Thanks!!
--
B4U
