Hi,
I wouldn't call this a standard procedure - generally speaking the most important two parameters you have to consider are: the protein/ligand affinity and the supply of the ligand. For tightly bound ligands, you may be able to just add the ligand once (i.e. during cell growth and expression, or upon lysis) thus avoiding the need to add the ligand to the chromatographic buffers throughout the process. This is perfect for ligands that aren't available in large quantities. The flip side of this is that when you're done purifying you're kind of stuck with the ligand you have - unless you successfully exchange it for something else (which is often hard to do completely if ligand has single digint nanomolar Ki or lower). On the other end of the spectrum are ligands that bind not as tightly and can be readily exchanged - these require you to have the ligand everywhere in the chromatography in the amount sufficient to elicit nearly full occupancy of the active site. Obviously it's better if you have such ligands in good supply. Examples of the tight-ligand co-purification are: GPCRs, nuclear hormone receptors (PPAR/RXR, RAR/RXR, etc.) - these proteins often require ligands for stability; and certain kinases that are otherwise toxic to the cells (PKC family) and have to be expressed/purified with a ligand because otherwise they kill the expression host. Examples of the medium- or weakly- bound ligand co-purifications include many nucleotide/side acting enzymes, aminotransferases (often need PLP to remain active and stable), proteins that require specific metal ions to remain in good shape, and a number of proteases that tend to chew themselves up unless they have an inhibitor to gnaw on. Sometimes ligands are co-purified unintentionally (famous example - quorum sensing factor with the crazy boron chemical) and pop up in the electron density as a surprise to the crystallographer. Please don't hesitate to ask specific questions :-) Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _____ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of protein.chemist protein.chemist Sent: Thursday, February 26, 2009 5:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Purification with ligand Hello, I wanted to know if there is a standard procedure for purification of protein with ligand. I have never done this before so it will be nice to get some help. Thanks, Mariah -- Mariah Jones Department of Biochemistry University of Florida
