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
