Hi Katherine, We had a case where we used saturating amounts of NaCl and precipitated the protein to get rid of a very tight binding ligand (Structure, 11, 677-690, 2003; look at the "preparation of the apoenzyme" section).
Regards, Mathews -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of SIPPEL,KATHERINE H Sent: Thursday, October 07, 2010 6:05 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Removing a tight binding ligand Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
