my comment on expression of protein being low and nonspecific binding. Amount of protein of expressed protein less = less resin = less nonspecific binding? (one have to do experiments to find right amount of resin to get least non-specific binding still pull out most of your protein of interest is a good idea?)
the original question was how can one do oncolumn digestion and will TEV bind to resin. Yes it will if it have tag. alternate source for TEV see www.mobitec.com MoBiTec GmbH, sell a TEV with additional GST tag. But if your protein is 87 and TEV should be possible to separate on GFC and you should add a GFC right after your TEV digestion which is always a good thing to do. earlier suggested NEB NiCo21(DE3) was promised to be useful but the benefit of this host system is still not clear to my experience. Hope this helps Padayatti On Thu, Jan 19, 2012 at 7:21 AM, Artem Evdokimov <artem.evdoki...@gmail.com> wrote: > Your mileage will vary: prset is an almost always quasi constituitive vector > due to leakage, with additional induction upon expression of T7 pol. This is > not always bad since some proteins do not fold well upon avalanche inducton > but do in fact fold when expressed at a trickle (even good old lac promoter > sometimes works better than T7). Due to leaky expression toxic proteins > usually have issues with prset, and often result in microcolonies, variable > colonies, loss of expression etc etc. It is not my first, nor my second > choice for an expression vehicle but it is something that is in my opinion > worth trying if one finds oneself in a tight corner. Either that, or call > Ghostbusters! > > Artem > > On Jan 19, 2012 5:19 AM, "Mark J van Raaij" <mjvanra...@cnb.csic.es> wrote: >> >> 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 -- Pius S Padayatti,PhD, Phone: 216-658-4528
