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A better way to His-tag

I would like to report a significant drawback of the 6x polyhistidine (His) tag for affinity chromatography.

Paul Riley(priley@temple.edu)
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In response to your article titled "Tag! Purifying Proteins with Affinity Chromatography,"1 I would like to report a significant drawback of the 6x polyhistidine (His) tag for affinity chromatography.

After purifying my 6x His-tagged homodimeric protein with Nichel nitrilotriacetic (Ni-NTA) resin, there were several doublets, or peaks of equal intensity in my two-dimensional nuclear magnetic resonance (NMR) heteronuclear single quantum correlation (HSQC) data. Since this suggested that my 20 kD homodimeric protein contained a mixed population of monomers, I was understandably worried. I tried cleaving my His-tag away before NMR, which was very laborious and expensive due to the protein quantities needed for NMR, but the doublets still remained.

When I tried purifying my protein from bacterial inclusion bodies and gel filtration, without Ni-NTA, the doublets disappeared, suggesting that the Ni-NTA resin had some effect on the structure of my protein. This suspicion was confirmed by a technical paper...

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