His-tagged proteins with less than six His

Per Mygind perm at medmicro.aau.dk
Sun Oct 18 03:30:33 EST 1998


John Philo wrote:

> Georg Wille wrote:
> >
> > Dear Bionetters,
> >
> > I'm about to attach an His-tag to the N-Terminus of a protein. Has anyone
> > ever heard of using _less_ than six residues successfully. I would like to
> > use as few as possible in order not to distort the enzymatic properties of
> > the protein too much and shifting its pI as little as possible.
> >
> > The protein is a dimer and its N-termini are fairly close together, so maybe
> > three His at each N-Terminus are maybe almost as good as six.
> >
> > Any experience? I would appreciate suggestions! Or maybe a reference to read
> > about this method.
> >
> > Thanks a lot!
> >
> >      Georg
>
> Georg,
>
> What the companies who sell the His-tag reagents fail to tell people is
> that in order to tight, specific binding your protein needs to be able
> to simultaneously interact with TWO metals and two of the NTA ligands.
>

Interestingly, is there any reference to this ?

>
> For this reason, and because the His tag is not always fully exposed,
> some labs have gone to using 7 or 8 His tags.
>
> You also need to remember that as a general rule His tag purification
> only works well under denaturing conditions.  Your idea that 3 His will
> work because your protein is a native dimer obviously only applies if
> you have a native folded dimer.
>

No, not in general. If your native purification fails, it might be because your
tag is puried in your protein, not being fully exposed.
To circumvent this, try moving the histag to the opposite end of your protein.
Imidazalo does not work so well in my hands.
Try washing in 1 M tris-hcl and eluting with histidines or EDTA (works perfect in
my experience using sixmer histidine tag)

>
> You are right, though, that the His tag can alter the properties of your
> protein, and we have seen examples of this.  One way around that is to
> engineer in a target sequence for a specific protease between the His
> tag and the N-terminus of your protein, and then cleave off the tag.
>
> 'Hope this helps.
>
> John Philo, Alliance Protein Laboratories
>
> *** Remove "*NO SPAM12*" from return address before replying. **

>
>
>
Regards

>
> Per Mygind
>
> ************************************************************************
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>
> Per Mygind, Cand.scient
>
> Department of Medical Microbiology and Immunology
> The Bartholin Building, University of Aarhus, Denmark
> phone : 89 42 17 47, fax   : 86 19 61 28
>
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