His-Tag:Single column prep

[Tracy Norris]

There has been some discussion recently concerning the use of His-tagged 
proteins for a single column prep. I have been using the His-vectors and 
Nickel resin from Qiagen for the purification of a several proteins,  
and have successfully purified them with this single step. Also, I have 
overexpressed all of these proteins in E.coli, and have only experienced 
a significant amount of background protein binding in one case (when the 
prep is done under denaturing conditions.)

However, I DO have another problem/question concerning His-tag 
purification. I am trying to purify a protein with a C-terminal 6xHis 
tag. I first tried an N-terminal tag, but it does not overexpress well 
and the N term has the domain of functional interest for the interaction 
I hope to see. The problem with the C-terminal tag is that the protein 
does not stick to the column unless the prep is done under denaturing 
conditions. I would rather do the prep under non-denaturing conditions 
in order to avoid the headache of refolding. So...I am considering 
trying a longer His-tag. Novagen sells a vector which puts on a 10xHis 
tag. My question is, has anyone had an experience where switching to a 
longer His tag (6 to 10) improved the binding to the resin? It would 
seem to me that the reason I am not seeing any binding is that the His 
tag at the C terminus is burried in the native state, and adding another 
4 His residues probably wouldn't really make any difference. But if 
anyone can give me evidence other wise I may give it a try. 

thanks,
Tracy Norris
tnorris at molbio.uoregon.edu