His-tags and DNA-binding

Slawomir Dabrowski 18-22 slawek at chem.pg.gda.pl
Fri Jan 12 04:24:52 EST 2001


Hi, I'm working with his-tagged SSB protein, and I've found that His
domain interrupted with ssDNA. I've found that His-tagged Taq DNA
polymerase has also activity problems.
Best regards,
SLawek


On Thu, 11 Jan 2001, Arnoud van
Vliet
wrote:

> > 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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