I don't know the exact reaction your protein is performing, but an
alternative would be to try to turnover your threonine substrate by
adding the other substrates. The reaction product should more easily
leave the active site. You might have to do an additional gelfiltration
to get rid of excess cosubstrates.
Good luck as well,
Herman
________________________________
From: CCP4 bulletin board [mailto:[email protected]] On
Behalf Of Richard M Salmon
Sent: Tuesday, June 26, 2012 1:06 PM
To: [email protected]
Subject: Re: [ccp4bb] practical protocol to remove cell culture
derived Threonine from the protein
Hi Wenhua
Have you tried using 6-8M urea or guanidinium hydrochloride to
denature your protein+threonine, then refold by dialysis into a
non-threonine-containing buffer? If you are performing ITC, then i guess
you already have an efficient activity assay, thus maybe use that as an
indicator of successful refolding....combined with successful crystal
formation may give you confidence in a correct, uniform fold too.
If your unfolded protein is His-tagged etc and dialysis isn't
totally doing the trick, then perhaps immobilising your protein to a
His-column might allow you to physically wash away the contaminating
threonine. You can then try an on-column refold, or elute and perform
dialysis refolding again.
Good luck,
Rick Salmon
On 26 June 2012 10:08, Wenhua Zhang <[email protected]>
wrote:
Dear ALL,
The threonine is found in the active site of my
protein structure from the crystallization in the absence of any
threonine containing chemicals. I presume that's why there was no
signals detected with the ITC experiment in which I titrate my protein
with Threonine. In the in vitro biochemical assay, threonine is one of
the substrates in the reconsitituted system.
So could anyone offer me a practical protocol to
remove the threonine from the protein for further experiment to confirm
the binding of Threonine to my protein by ITC.
Thank you all.
Wenhua
Ph. D student in structural biology
Paris-Sud XI, France
