Dear Stefano, Here few thoughts: You should calculate the amount of holo/apo protein that you get after the column. A ratio 280/450 between 9-12 it will suggest that your protein is almost completely in the holo-form. So, I will be worried about the peak of flavin only if this ratio become much higher than 12. If this is the case, I would analyze the flavin peak to evaluate if it is really FAD or, for example, FMN. You can do easily with a quick silica TLC. This mainly because can happen that during the folding there is a misincorporation of the cofactor (I am assuming that you are sure that FAD is your cofactor). Otherwise, if your expression levels are too high, maybe the expression strain is not able to produce enough flavins to be incorporated. In this scenario, you can try to slow down the induction and/or use more rich media (like terrific broth, where the excess of glycerol can also help in keeping bounded the flavin) as add 1-2 mM of riboflavin in the broth.
If this doesn't work, during the purification I would suggest to add some FAD in the lysis buffer (~50 uM) and 10% glycerol to increase the viscosity of all the buffers and reduce the diffusion. You would maybe need also to play with salt concentrations to see if there is an effect in the flavin binding. Remember: flavins are also light sensible. Be sure to keep the sample in the dark as much as possible during all the steps (i.e. use aluminum foil to wrap the column or the tubes where your sample is stored). To remove FAD there are many protocols that you can follow, but mainly it require a dialysis in presence of KBr in a light acidic environment. You can find protocols in literature looking for papers by Edmondson, Van Berkel or Massey to start or chapter 11 in flavoprotein protocols (Vol 131, methods in molecular biology). Good luck, Roberto __________________________________ Roberto Orru, PhD Department of Chemistry, Emory University 1515 Dickey Drive Atlanta, GA. 30322 (USA) -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Benini Stefano (P) Sent: 13 March, 2014 18:40 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic: protein losing FAD during purification 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." ________________________________ This e-mail message (including any attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. If the reader of this message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this message (including any attachments) is strictly prohibited. If you have received this message in error, please contact the sender by reply e-mail message and destroy all copies of the original message (including attachments).
