Re: [ccp4bb] off-topic: protein losing FAD during purification
Hi Stefano, On top of all that has been suggested you should also be aware of the effect of pH and buffer composition on the apo-holoenzyme equilibrium during purification and crystallization. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Benini Stefano (P) [stefano.ben...@unibz.it] Sent: Friday, March 14, 2014 12:40 AM 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.
Re: [ccp4bb] off-topic: protein losing FAD during purification
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) 353-9334Email: rmgarav...@gmail.com On Mar 13, 2014, at 6:40 PM, Benini Stefano (P) stefano.ben...@unibz.it 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.
Re: [ccp4bb] off-topic: protein losing FAD during purification
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).
Re: [ccp4bb] off-topic: protein losing FAD during purification
Dear All On similar lines any suggestions and tips on expression and purification of the proteins containing both FAD and NADPH would be really helpful. I recently tried to express and purify a protein containing these two ligands, but failed (aggregation of protein). my initial questions are: does addition of Riboflavin in the media has affect on protein expression? considering reconstitution, I am concerned that NADHP will be oxidized during processing. what are the other alternatives for incorporating ligands, if reconstitution doesn't help?
[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.
Re: [ccp4bb] off-topic: protein losing FAD during purification
Hi Stefano, Wouldn't you be better off going the other way around, that is supplementing your protein solution with FAD prior to setting up the crystallization trials? It sounds as if the FAD is quite labile in your case, but it could well stabilize the protein and hence give rise to better diffracting crystals. Unless of course you have good reason to believe that the apoprotein is more stable, in which case dialysis could be the way to go in order to get all (??) the FAD out. My 2p thoughts. Good luck, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Benini Stefano (P) [stefano.ben...@unibz.it] Sent: Friday, March 14, 2014 12:40 AM 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.
Re: [ccp4bb] off-topic: protein losing FAD during purification
In cases like this, I keep FAD around during all steps starting from cell lysis, purification, dialysis and even crystallization. In one case we added FAD to protein such that the protein: FAD ratio was 1 prior to setting up crystallization trials. Hope that helps. Harkewal Sent from my iPad On Mar 13, 2014, at 5:40 PM, Benini Stefano (P) stefano.ben...@unibz.it 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.
Re: [ccp4bb] off-topic: protein losing FAD during purification
And include FAD at a few uM in your column buffers. Although if you are getting two separate sharp peaks for protein and FAD, it doesn't sound like it is bleeding off during chromatography but rather already dissociated in the material you apply to the column. Take a spectrum of the material in the peak you suspect of being FAD- Free FAD has a huge peak at 260 and smaller peaks around 375 and 450. Boaz Shaanan wrote: Hi Stefano, Wouldn't you be better off going the other way around, that is supplementing your protein solution with FAD prior to setting up the crystallization trials? It sounds as if the FAD is quite labile in your case, but it could well stabilize the protein and hence give rise to better diffracting crystals. Unless of course you have good reason to believe that the apoprotein is more stable, in which case dialysis could be the way to go in order to get all (??) the FAD out. My 2p thoughts. Good luck, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Benini Stefano (P) [stefano.ben...@unibz.it] Sent: Friday, March 14, 2014 12:40 AM 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.
Re: [ccp4bb] off-topic: protein losing FAD during purification
Dr. Stefano, 1) I would add FAD in all buffers after lysis,even after GF you could perform dialysis with buffer supplemented with FAD. 2)for the second question I found this paper which could be helpful: Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography. Good luck, Mirella Sent from my iPhone On 13 Mar 2014, at 22:40, Benini Stefano (P) stefano.ben...@unibz.itmailto:stefano.ben...@unibz.it 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.
