Does His-tag have to be terminal?

Nikolai Chitaev nikolaic at VISAR.WUSTL.EDU
Fri Jan 19 12:50:52 EST 1996


> To: methods at net.bio.net
> From: wind at biobase.dk (Troels Wind)
> Subject: Does His-tag have to be terminal?
> Date: 18 Jan 1996 13:13:38 GMT

> Hi all,
> 
> I'm currently planning to modify an expression vector by introducing a
> His-tag for easy purification of recombinant protein.
> 
> The vector as it is fuses a C-terminal immunotag (c-myg: EQKLISEEDLN) to
> the protein, and the easiest place to put the His-tag is between the
> protein and c-mug, resulting in something likes this:
> 
> N--/protein/--6xHis--/c-myg/--C
> 
> My question is: Will the His-tag still be functional when it is not at
> the extreme terminal of the protein?
> 
> I know N-terminal fusion would be a solution, but it's not possible in
> this context. Furthermore, I realise that much of the answer to my question
> depends on folding, which is rather inpredictable, but I would like to hear
> if anybody had tried something like this before.
> 
> Thanks in advance,
> 
> Troels Wind
> Institute of Molecular and Structural Biology
> University of Aarhus
> Denmark
> 
> 

Dear Troelis.
As far as I understand from your post, you need both the 6-his tag and Myc tag. 
Using anti-Myc antibodies you could perform a functional assays with your 
protein, but 6-his tag will help you to purify the recombinant protein. Is the 
above assumption correct?
If its correct then you should avoid using Myc at extrem C-terminus, because 
doing so you are placing purification tag upstream of your functional tag. 
Since C-terminal portion of the protein is eukariotic (and due to a lot of 
other causes), you`ll get a lot of your rec.protein which will have 6-his tag 
but lack Myc tag. You`ll succecfuly purify the suff which you don`t need, and 
for you there will be no (exept affinity column) way to separate wanted 
product, from this type of contaminant. Try to place 6-his tag at extrem 
C-terminus.

Nikolai
http://128.252.119.253






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