6X His Tag vector construction assistance needed

[pedrochang]

Hi Everybody,

New to the forum. First time post, so greetings.

I am designing a vector that starts like this :

( backbone - ATG -Signal sequence - MCS - gene - MCS - TGA - backbone )

and i want to make

( backbone - ATG -Signal sequence - 6xHis - MCS - gene - MCS - TGA - backbone )

my question to all you gurus out there,

when the signal sequence is clipped and the protein is secreted, will it be a problem if there are a few odd amino acids (because of the MCS) before the 6XHis? Or must the 6XHis be strictly at the beginning of the clipped/secreted protein?

Or is a N-terminal 6X His impossible?

Much thanks in advance for your reply.

[snowcapk]

I think it depends. If you are using Qiagen's NI-NTA system (or some other nickel or cobalt-based system) to purify the His-tagged protein, it shouldn't matter for that step whether there are a few extra aas at the N terminus before the 6xHis tag.

However, you may have a problem if you try to remove your 6xHis tag later, depending on whether you use an exo- or endopeptidase. The endopeptidase should work fine, but if you use an exopeptidase and there is a "stop residue" for it before your 6xHis tag, then your His tag won't be removed. Your supplier should tell you what the stop residues are; if you use Qiagen's TAGZyme system, they have a web applet where you can check your N-terminal sequence for stop residues. (http://www1.qiagen.com/products/protein ... fault.aspx)

Another problem could occur if you will use anti-6xHis antibodies to detect or purify your protein. It may be that the binding of the antibody is influenced by the peptide sequence surrounding the 6xHis tag. I know that these antibodies are usually successful at recognizing an MHHHHHH.... epitope, but I don't know if they would recognize MWRKRKRHHHHH, for example. I would contact the antibody supplier because it seems like this might vary.

[pedrochang]

hi snowcapk

Sorry for the delay. Thanks very much for your explanation. Was hyper-paranoid and decided to use quickchange to insert the 6X His between gene of interest and the signal sequence. Hope it remains 6X. Thanks again.

pedro