Another option is to try NEB NiCo21(DE3) cells.
http://www.neb.com/nebecomm/products/productC2529.asp
I have no relation to NEB beyond being a customer.
They've mutated GlmS to eliminate binding to IMAC resins and have
added chitin affinity tags to SlyD, ArnA and Can to allow simple post-
IMAC removal of those common contaminants.
I've just started testing them and would be interested in feedback
from others.
Best,
Cynthia
On Jan 18, 2012, at 10:21 AM, Ed Pozharski wrote:
On Wed, 2012-01-18 at 18:26 +0530, PULSARSTRIAN wrote:
The Problem is I am not able to get rid of the infamous contamination
proteins of arnA gene (72 kDa) and glmS gene (67 kDa).
This is only a problem if you plan to have imac purification as your
only step. If the goal is crystallization, such products can
definitely
be used in initial trials (in fact, impurities may provide a benefit
of
nucleation), but you would have to introduce a second step eventually,
which is often the ion-exchange. The co-purified proteins will be
definitely removed at that step, so perhaps your time is better spent
optimizing some high-res ion-exchange gradient.
Two ways I can think of if you'd rather stick with IMAC is to try
cobalt
instead of nickel (might have different non-specific binding
profile) or
overload the resin with your protein (the presumption here is that it
has higher affinity than impurities and you will get rid of them by
simple competition). Or maybe the imidazole gradient could help.
Cheers,
Ed.
--
"I'd jump in myself, if I weren't so good at whistling."
Julian, King of Lemurs
