How can I remove the Polyhistindine tag or myc epitope from my recombinant prote - (Apr/06/2006 )

Dear all,

I want to remove the polyhistidine tag or myc epitope form the recombinant proteins(my recombinant protein has myc epitope and polyhistidine tag in C-terminal). Any idea????

Many Thx!!!!!

siuchi

Have you already expressed your protein, our do you still have to do so?

There has to be a cleavage site between your protein and the tags. If there isn't one, you can clone one into your expression vector. I've cloned a TEV site into one of my vectors for this purpose. If you've already produced the protein, and there is no cleavage site, then you're out of luck.

Dear jaknight,

Thank you for your kind reply!!! Could you explain more detail how can i clone a TEV site the vector? What enzyme should be used to cut the tag? thanks very much!!!!

siuchi

No problem.

The enzyme to use is called TEV protease (Tobacco Etch Virus protease). You can buy it from Invitrogen.

The protease recognises the peptide sequence ENLYFQG, cleaving between the Q and G.

So you would want to clone in the appropriate dna sequence between your insert and the tag. I ordered two oligos, each 21bp in length plus the appropriate overhangs, annealed them together, phosphorylated them with T4 kinase, then ligated them into my cut vector. It was pretty straightforward.