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 >
