Do we need to place the insert in a specific site in his tagg vector for further protein purification or can we just use any restriction site present in the vector?
Dear Abire
this generally depends from the sequence of your vector.
It may happen that the selection of different enzime can produce the His-tagged, untagged or Hiss tagged plus recogniction site of a protease as Fatt.XA or REK or TEV.
To give you more detail i need to know better your vector.
However you can see some example of different constructs that you can obrain selecting different enxime couple in pet16b in the presentation:
ProteoCool n°2 (Primer Design: Restiction/Ligation) avialable on my blog:
PRoteoCoool https://proteocool.blogspot.com/ at page1: Cloning
i hope that it can be usefull to clarify some of your doubts
best regards
Manuele
There is no general answer to your question. In the absence of further knowledge, I prefer to choose a vector that generates a polypeptide comprising an N-terminal His-tag, a Tev protease processing site and the sequenced encoded by the insert. Thus, if need be, the His-tag can be removed from the affinity-purified polypeptide, leaving the remaining sequence with only one or two extra amino acid residues. However, in some cases it may be necessary to fuse the His tag at the COOH end, e.g. if the NH2 fusion causes the target polypeptide to be insoluble, or if the NH2 terminus lies in such a recessed site that the tag is no longer accessible for affinity purification. In that case, there is not much purpose in adding a Tev site, because Tev cleavage will still leave five extra residues, ENLFY, after cleavage
Thankyou all, I want to use pet32 vector to clone first with mcherry and then with my gene of interest for further protein purification, but i'm wondering where to place these two for convenient protein purification as I also want the fluorescent mcherry to visualize at microscope.
- Badges
- Science method