Does His-tag have to be terminal?

[davesmith at bioch.tamu.edu]

wind at biobase.dk (Troels Wind) wrote:


>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

Troels,
	I don't know if this is what you are looking for or not, but we work
with an integral membrane protein which we believe is bitopic with a
very short, but "soluble" loop in the periplasm.  We have his-tags at
the N- and C- termini; N-teminal is partially functional; C-terminal
is partially functional, but with a different phenotype.  We also put
a his tag in the loop of the protein with Gly on each side of the tag.
It is almost indistiguishable from wild-type with respect to
phenotype.  If I were you, I'd sure give your idea a try...you might
consider a couple of flanking "flexible" residues when you make it.
Good Luck!
Dave.