Have you tried reverse purification over the Ni-NTA column? That is the typical next step in purification after His-tag cleavage. Or did you mean to say that the impurities elute off with your cleaved protein during the reverse purification? If this is the case, you could try adding a reducing agent to help prevent unwanted interactions. You could also try a subsequent purification using ion exchange. As for on-column digestion... it is possible but comparatively inefficient. Hope this helps.

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Greg Costakes
PhD Candidate
Department of Structural Biology
Purdue University
Hockmeyer Hall, Room 320
240 S. Martin Jischke Drive, West Lafayette, IN 47907

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** Hard work often pays of in time, but Procrastination always pays off now **

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From: "PULSARSTRIAN" <[log in to unmask]>
To: [log in to unmask]
Sent: Wednesday, January 18, 2012 7:56:39 AM
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!!



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B4U