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.
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.
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
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