His tagging and 'cheaper' Nickel columns

Curt Ashendel ashendel at aclcb.purdue.edu
Thu Nov 16 10:24:01 EST 1995


On Thu, 16 Nov 1995 07:38:15 GMT, 
Richard A. Jefferson  <raj at cambia.org.au > wrote:

>We are using His-6 tagging to purify many variants of mutagenized proteins for 
>analysis, and are in need of an inexpensive (relatively) method to do so.   
>The columns and column resins sold by Qiagen (Ni-NTA), Clontech (TALON), 
>Invitrogen (Probond), tend to be very expensive.
>Can someone recommend either an effective 'home-made' version of the Nickel 
>chelate methods (and a source for the materials), or give an assessment of the 
>relative merits of the above resins, and their re-useability?  

My comments are limited to home-made NTA-agarose:

I have successfully synthesized the NTA ligand and coupled it to 
agarose (Sepharose CL-4B). It is relatively inexpensive and fairly 
easy to do, though a somewhat specialized set up is needed to do the 
catalytic hydrogenation deprotection (second step). The 
sythesis and coupling using epibromohydrin is described 
by Hochuli, Dobeli, and Schacher in J. Chromatography 411: 177-184 
(1987). Basically, CBZ-epsilon-lysine (available from Sigma Chemical, 
Aldrich Chemical and many other suppliers for US$2.40 to 3.40/g, and 
only 3 to 4 g are needed to make enough NTA ligand to couple to 100 ml 
of resin) is reacted with one equivalent of bromoacetic acid (very 
inexpensive) in 2M NaOH. The protected product is isolated by 
crystallization from HCl and is deprotected by catalytic hydrogenation 
(on Pd-C). The product is pure after filtering off the catlyst and 
drying the residue, though drying is not needed except to determine 
yield. Though this sounds a bit intimidating, it is not at all 
difficult and is very clean chemistry. If done on the 100 to 300 mmole 
scale, you have enough to make a lot of resin (20 mmole/100 ml resin). 
The Sepharose is the most expensive component of the preparation.

I have also coupled the ligand to sepharose using epoxy activation 
with 1,4-butanediol diglycidyl ether according to Porath and Axen, 
Meth. Enz. 44: 19-45. This seems to work just as well for Ni-chelate 
chromatography.


Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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