avoid pRSET would be my recommendation - my impression from a couple of examples is pRSET gives badly folded protein.
Can you easily subclone it in a better expression vector?
(personally, even if it were not so easy, I would reclone it in another expression vector).
Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij
On 18 Jan 2012, at 13:56, PULSARSTRIAN wrote:
> 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
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