Stefano, Before you address the problem, you need to ask yourself a couple of things.
You say that "on the gel filtration we clearly see two bands corresponding to holoprotein and free FAD." That is not too odd, but have you ask the question is all the protein good protein. Is this an enzyme? If so, assay samples before and after the gel filtration column without adding extra FAD and compare to assays with a slight excess FAD. If the specific activities are different (slight excess FAD > before gel filtration & no added FAD > after the gel filtration & no added FAD), I would follow the good advice of the others. You have just removed exchangeable FAD and are crystallizing a mixed system. Is this a recombinant protein partially purified using a His-tag or such? If the specific activities are the same, I would suspect that not all the protein is in good shape. Try ion exchange chromatography to see if two different protein variants can be separated. With luck that can be (1) active holoprotein and "poorly-folded" protein (always a problem with His-tag purified recombinant protein) or (2) active holoprotein and apo-protein. If it is not an enzyme, or it is not an easy assay, take the spectrum of the gel filtration-purified holoprotein and see if it differs from free FAD, then add FAD to a near stoichiometric level to see if you can saturate without a spectral change (e.g., a blue-shift of the absorption peak). That could tell you if it is an active holoprotein and apo-protein problem or active holoprotein and "poorly-folded" protein problem. Sometimes "poorly-folded" protein weakly binds the cofactors, but not in the native way. If you are lucky, supplementing with FAD, either before, during, or after purification will work fine. In some cases, I have found that it is better to purify the apo-protein, then reconstitute the holo-protein, if the former is stable enough. However, some heme, NAD(P)(H), and FAD binding protein allow removal of the cofactors but not its reconstitution. It depends are your protein system. A little more biochemistry is needed, but find a nice assay system to verify what you are doing. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected] **************************************************************** On Mar 13, 2014, at 6:40 PM, Benini Stefano (P) <[email protected]> wrote: > Dear All (those dealing with wetlab stuff..), > > While purifying a FAD containing protein we lose part of the FAD (on the gel > filtration we clearly see two bands corresponding to holoprotein and free > FAD). > > We obtain crystals but diffracting to only about 4 A despite their beautiful > look. Our hypothesis is that the crystals contain a population of molecules > with and without FAD (?). > > The questions are: > > 1) how to keep FAD bound to the protein during purification and > crystallization? > > 2) how to completely remove FAD from the protein? > > Thank you very much for any help provided! > > Best regards > > Stefano (part-time wetlab person) > > > Dr Stefano Benini, Ph.D. > Assistant Professor > > First International workshop: "Molecular Basis of Fire Blight", Bolzano > 15.10.2014 > > Laboratory homepage: > http://pro.unibz.it/staff2/sbenini/B2Cl.htm > > Personal homepage > http://pro.unibz.it/staff2/sbenini/ > > "I don't like anything that's fake and I hate pretenders!" > > ********************************************* > Bioorganic chemistry and Bio-Crystallography laboratory (B2Cl) > Faculty of Science and Technology > Free University of Bolzano > Piazza Università, 5 > 39100 Bolzano, Italy > Office (room K2.14): +39 0471 017128 > Laboratory (room E.021): +39 0471 017910 > Fax: +39 0471 017009 > ******************************************** > "ogni giorno in più è un giorno in meno."
