[ccp4bb] metal chelation
Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
Are you treating all your buffers with metal chelating resin? Are you washing all plasticware with EDTA and metal-free buffer prior to use? How are you quantifying your metal content, and what metal ions are contaminating your sample? You might be pulling out the metal ions, but they get right back in as soon as you remove the chelators. Metal ions are present at trace levels in all water and chemicals (Zn is particularly common). You might need to 'soften up' the protein a little bit, we use a pH 3.8 stripping protocol. Denaturants may be required. Time is also a factor, extending your chelating step might help. It's not trivial to make app-enzymes, in my experience, Nat On Mon, May 19, 2014 at 12:20 PM, SUBSCRIBE CCP4BB Adam Brummett adam-brumm...@uiowa.edu wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Adam, - - many plasmids for His-tags contain a cleavage site to remove the tag. In fact this provides you with an additional purifiction step which is complementary to the first Ni-column and therefore quite a good strategy (in addition to chopping off the chelating His-tag). - - you can try other Titriplexes like EGTA, EDTA is only one of a series - - if only the His-tag binds the ion, wouldn't this qualify the protein as 'apo'? If the His-tag is ordered, you could actually replace the ion with Co and use this for phasing, in case this is an issue. Best, Tim On 05/19/2014 07:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTekO/UxlJ7aRr7hoRAkhUAKCXbpCx2x4arUbTDtnpDdviIoL3/QCeP+ik aaDa+rmp1AALJhdsDn8els0= =hfzc -END PGP SIGNATURE-
Re: [ccp4bb] metal chelation
The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are not going to give up their metal to chelators, or just any chelator, or at all. Denaturation, dialysis, and refolding is an extreme way of removing metal ions to make apoprotein. Won't work for every protein. Chelation can be highly specific, that is one chelator may work, while another, similar one, will not. Some metal ions are notoriously difficult to eliminate, because they are adventitious trace contaminants in nearly everything, e.g. zinc and maybe even iron. (Plastic-ware seems to be often loaded with trace iron, and also is capable of adsorbing metal ions form solution.) To make apo-enzymes from zinc proteins, you have to go to heroic efforts to ensure that glassware, water, buffers, and reagents are zinc-free, especially if you don't have high (mM) concentrations of protein to work with. A His-tag is very likely to snag adventitious metals from solution, and can often mess up metal analysis for metalloproteins by providing extra metal. If this is a problem for your application, you may want to consider removing the His-tag. If you are making apoenzyme to get a different metal installed (metallosubstitution), there are slightly easier ways to do that than going through the apoenzyme route. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett rrowl...@colgate.edu wrote: The answer depends on a number of questions: What metal ion are you trying to eliminate? What kind of metal-binding site is involved? A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are not going to give up their metal to chelators, or just any chelator, or at all. Denaturation, dialysis, and refolding is an extreme way of removing metal ions to make apoprotein. Won't work for every protein. Chelation can be highly specific, that is one chelator may work, while another, similar one, will not. Some metal ions are notoriously difficult to eliminate, because they are adventitious trace contaminants in nearly everything, e.g. zinc and maybe even iron. (Plastic-ware seems to be often loaded with trace iron, and also is capable of adsorbing metal ions form solution.) To make apo-enzymes from zinc proteins, you have to go to heroic efforts to ensure that glassware, water, buffers, and reagents are zinc-free, especially if you don't have high (mM) concentrations of protein to work with. A His-tag is very likely to snag adventitious metals from solution, and can often mess up metal analysis for metalloproteins by providing extra metal. If this is a problem for your application, you may want to consider removing the His-tag. If you are making apoenzyme to get a different metal installed (metallosubstitution), there are slightly easier ways to do that than going through the apoenzyme route. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
Hi Adam, I have not read all the thread as it came all at once and late (9:00pm here). I believe the best way to strip a protein of metals is to first adsorb it onto a solid support (e.g. IEX) and then use a sufficiently low-pH (say equal or below 6) buffer that contains also EDTA. You will probably need several washes but it works! Also be aware that EDTA binds well to several proteins. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies 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.Mrabet at univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 19/05/2014 20:21, Adam Brummett wrote: Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu wrote: The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are not going to give up their metal to chelators, or just any chelator, or at all. Denaturation, dialysis, and refolding is an extreme way of removing metal ions to make apoprotein. Won't work for every protein. Chelation can be highly specific, that is one chelator may work, while another, similar one, will not. Some metal ions are notoriously difficult to eliminate, because they are adventitious trace contaminants in nearly everything, e.g. zinc and maybe even iron. (Plastic-ware seems to be often loaded with trace iron, and also is capable of adsorbing metal ions form solution.) To make apo-enzymes from zinc proteins, you have to go to heroic efforts to ensure that glassware, water, buffers, and reagents are zinc-free, especially if you don't have high (mM) concentrations of protein to work with. A His-tag is very likely to snag adventitious metals from solution, and can often mess up metal analysis for metalloproteins by providing extra metal. If this is a problem for your application, you may want to consider removing the His-tag. If you are making apoenzyme to get a different metal installed (metallosubstitution), there are slightly easier ways to do that than going through the apoenzyme route. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
Adam, We developed a protocol (loosely based on a few previous literature reports) for metallo-substitution of beta-carbonic anhydrase (a zinc-metalloenzyme) with cobalt(II). The metal ion in this enzyme is extremely refractory toward extraction with chelators, and the protein will not denature/refold at all. Briefly, our protocol involves overexpressing protein in minimal media in the presence of 10-100 uM Co(II) ion. This allows us nearly 100% metallosubstitution of Co(II) for Zn(II) during the overexpression phase. We have verified that our commercially prepared minimal media is quite metal-free. The protocol is described here: * Katherine M. Hoffmann,† Dejan Samardzic,* Katherine van den Heever,* and Roger S. Rowlett§, “Co(II)-substituted Haemophilus influenzae β-Carbonic Anhydrase: Spectral Evidence for Allosteric Regulation by pH and Bicarbonate Ion,” Arch. Biochem. Biophys. 2011, 511, 80-87. A couple of tricks we discovered: * Thiamin supplementation is required for good growth in minimal media * We have to use conditioned plastic expression flasks for this to work well. Acid-washed flasks result in no cell growth. Flasks that have been used to do regular overexpression runs, but have been simply well rinsed out with deionized water work reproducibly well. I'm pretty sure that the walls of the conditioned flasks are providing sufficient trace metal ions for growth, without swamping out our supplemental Co(II) ion with contaminant ions. FWIW, every time we do ICP analysis of metalloenzymes that are His-tagged, we nearly always get non-stoichiometric extra metal ion. It's maddening when you are trying to establish protein:metal stoichiometry, or determine accurate protein concentrations this way. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 3:04 PM, Nadir T. Mrabet wrote: Hi Adam, I have not read all the thread as it came all at once and late (9:00pm here). I believe the best way to strip a protein of metals is to first adsorb it onto a solid support (e.g. IEX) and then use a sufficiently low-pH (say equal or below 6) buffer that contains also EDTA. You will probably need several washes but it works! Also be aware that EDTA binds well to several proteins. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies 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.Mrabet at univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 19/05/2014 20:21, Adam Brummett wrote: Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu wrote: The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are
