HELP: Purification of his-tagged protein

David Ackerley david at sanger.otago.ac.nz
Wed Nov 20 22:51:35 EST 1996


I'm a molecular biologist foolishly trying to enter the realms of protein 
purification, and encountering a few problems along the way. The 
his-tagged protein I'm working with is around 118kDa, and can (although 
not wonderfully clearly) be seen to appear after induction on SDSPAGE of 
crude cell extracts. However, after purification on a Gibco NiNTA resin, 
I am left with barely detectable amounts of my protein, not nearly enough 
for sequencing to confirm id. The only product I am managing to purify 
significant quantities of is the 70ish kDa E. coli GRO-EL protein (or 
something like that?) that is apparently commonly copurified in this type 
of procedure. I have tried the following protease inhibitors, both 
separately and together; PMSF, pefabloc, pepstatin, TPCK, and soybean TI. 
But no luck. Sigh...

	Anyhow, my question is this: has anyone out there encountered 
similar problems, and if so, what did you do about it? I am considering 
looking at other purification matrices, but thought I would see if anyone 
with more experience than myself (ie just about anyone) had any helpful 
(and hopefully cheaper!) suggestions to make. Any such suggestions will 
be welcomed!

	Cheers, David Ackerley

 



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