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
