His-tags and DNA-binding

Arnoud van Vliet avvliet at knoware.nl
Thu Jan 11 15:12:52 EST 2001


> Has anyone come across a situation where a 6XHis tag has affected the
> DNA-binding of a DNA-binding protein? (the pH is 8 so unlikely to be +ve
> charge). I know the folding could be affected. Just wondered if anyone
> had seen such a phenomenon...

Hi Matt,

I might have, but it was probably more indirect. I work with Fur, a
bacterial repressor which forms a complex of two Fur molucules and the
iron-cofactor. The N-terminus binds DNA, the C-terminus binds iron and
allows the dimerization.

When I expressed Fur with a N-terminal 6His-tag, the protein did not perform
properly in gelshifts. We think this is because we introduced a second
metal-binding domain with the 6His, totally screwing up proper folding or
dimerization or DNA-binding.

Not much of a help, I am afraid, but I hope it's useful to you.

best wishes
Arnoud







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