Re: [ccp4bb] Removing a tight binding ligand
We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine 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
Re: [ccp4bb] Removing a tight binding ligand
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
Re: [ccp4bb] Removing a tight binding ligand
Hi, It could help if you said what your ligand is. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabetat medecine.uhp-nancy.fr On 08/10/2010 03:05, SIPPEL,KATHERINE H wrote: 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
Re: [ccp4bb] Removing a tight binding ligand
It is just like the regular dialysis but the deserved buffer contains charcol powder. Meng-Chiao Joseph Ho How do you do this ? I have not heard of this, but I also never had to deal with getting rid of a ligand. However I would be interested to learn more about this method. Thanks, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 8, 2010, at 11:01 AM, Joseph Ho wrote: We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine 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
Re: [ccp4bb] Removing a tight binding ligand
I would like to thank all of you for your replies. I very much appreciate your time and I've got some great starting points to try out. I will put together a recap of the off- and on-board comments in the next day or so for archive posterity. For those looking for more details... The protein is specifically a monomeric substrate binding protein (very roughly similar to MBP) and binds a water-soluble vitamin. It's purified exclusively by ion exchange so no tags though it is shy a bit of the N-terminal domain to remove a lipo- moiety. I have no idea what the Kd is at the moment, that was what I was hoping to calculate. I do have the bound structure, it is not covalently attached to the protein. The interactions are mostly hydrophobic with only a couple of hydrogen bonds. Once again, thank you all, Katherine On Fri, Oct 8, 2010 at 12:20 PM, Joseph Ho j...@medusa.bioc.aecom.yu.edu wrote: It is just like the regular dialysis but the deserved buffer contains charcol powder. Meng-Chiao Joseph Ho How do you do this ? I have not heard of this, but I also never had to deal with getting rid of a ligand. However I would be interested to learn more about this method. Thanks, J??rgen - J??rgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 8, 2010, at 11:01 AM, Joseph Ho wrote: We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine 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 -- SIPPEL,KATHERINE H Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] Removing a tight binding ligand
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. I am assuming that the protein is tagged in someway and that you add your purified protein to Urea/Guanidine and refold by dialysis. If you protein is His-tagged, I would unfold it on the column and flush a large excess of Binding buffer (containing denaturant) to remove the ligand and elute the protein in elution buffer (containing denaturant) and then refold, to ensure that no ligand is present during refolding. If this is what you have done, ignore this. For the protein that cannot be refolding, have you investigated thermal denaturation. I have removed a 1kDa ligand by placing a nickel column in a water bath set near to the Tm of the protein. After flushing an excess of prewarmed buffer through, the water bath was switched off, allowed to cool to RT and then protein eluted off. Around ~95% was refolded. The unfolded protein was then separated by size-exclusion. Though I have no evidence for this and I am just thinking out aloud (please could someone correct me if I am wrong, have evidence to the contrary or both), if the ligand is not present in the periplasm, and the protein is targeted through the sec pathway (which recognizes unfolded proteins), purification from the periplasm could circumvent refolding if you are lucky and you do not get cytoplasmic contamination. All the best Dan
Re: [ccp4bb] Removing a tight binding ligand
Kd is crucial. If this is something like streptavidin and biotin, there is no way to separate them without denature the protein. You can try varying pH, binding it to ion exchange column and then washing it with large volume of buffer, as mentioned in MBP manual from NEB, or running it through a hydrophobic column after treating the protein with really high salt. The last one worked for one of my colleagues. Although I still doubt you can completely get rid of the ligands. Nian Huang, Ph,D. Dept. of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Thu, Oct 7, 2010 at 8:05 PM, SIPPEL,KATHERINE H ksip...@ufl.edu wrote: 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
