his tag - his tag purification (Apr/28/2005 )

Hi,
i want to add his tag to my protein, its a bacterial luciferase enzyme subunit and i don't know where active site is, should i put it N or C-terminal, and the expression vector to having low copy # plasmid and producing soluble protein, also which purification system is the best and are commercially packed columns better than lab packs, another thing is if i don't want to go for western blotting, what would be the size difference in tagged and non-tagged protein to detect it, last question is what should be the optimal length of the tag.
i'll greatly appreciate an early response.
thanks a lot
nbj

Hi All,
another thing i want to know is i need to use DTT in my buffers, would that affect Ni resin, as Amersham claims their resin is compatible with reducing agents, any experience ?
thanks
nbj

Hi Nbj
Try using a hexa histidine tag (his6). Since this is a small tag, normally it wont affect the activity of the protein. Use vectors like pRSET (Invitrogen) or pET.
We use Probond column (invitrogen) and it is giving good results for our recombinant protein purifications. The BD Talon resin (BD Clontech also gives good results).
Try using an overexpression system like pET coupled with BL21 DE3 strain of E.coli to maximise ur expression.
Add of the His6 tag will add an extra of ~1 Kda to ur protein size.

Regards
Avinash

=========================
Avinash
http://aviprem.gq.nu
-------------------------------------------------
To err is human, to moo is bovine.
==============================