Make sure the imidazole is removed before you apply the TEV digested sample back to the Ni column for the reverse Ni step.
Sounds like the expression level of your protein is low. You many consider to improve the expression. Those contamination proteins disappear from Ni elution when the target protein is abundant. As mentioned by Greg Costakes, on beads/column cleavage is less efficient comparing to solution digestion. However on column digestion is useful for some difficult-to-purify proteins. We have used our product TurboTEV (http://www.accelagen.com/TurboTEV.htm) for on column digestions. We found that keeping the TurboTEV to target protein ratio at 1:20 to 1:100 (same ratio for digestion in solution) gives good digestion, although never complete. That means you need to use >0.2 mg/ml TurboTEV for a resin with binding capacity of 20 mg/ml. We made TurboTEV storage buffer compatible with Ni resins. TurboTEV has dual GST- and His-tags. The geometry could be different from His-tagged TEV products. TurboTEV also has very high specific activity due to a novel stabilizing mechanism that is independent of S219 mutations. These properties probably make TurboTEV more efficient for on column digestion than other TEV products. You need to experiment a little bit with His-tagged TEV products. Chun Accelagen From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of PULSARSTRIAN Sent: Wednesday, January 18, 2012 4:57 AM To: [email protected] Subject: [ccp4bb] His Purification 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
