Depending on the zinc-binding site, it may not bind Fe(II) at all. Zn(II) and Fe(II) have very different preferred ligand binding environments. For many zinc-metalloenzymes, substitution with Fe(II) would be difficult to impossible. In general, you will find it very difficult to make your non-defined expression medium zinc-deficient. Zinc is a very common component of complex media, and is also a very common adventitious contaminant. Ideally, you will want to include the metal ion in the expression medium so that it can be incorporated at the time of protein synthesis. In many cases, this will enhance the stability of the synthesized protein. For bacterial overexpression at very high protein levels, 10-100 uM metal ion is more than plenty. More than that is actually toxic in bacterial systems, as it may impede critical iron transport into cells. But we have found that complex media already contains more than enough zinc to populate overexpressed proteins. We only supplement when we are trying to make metallosubstituted protein, in which case we use defined zinc-deficient media and supplement with a compatible metal ion (e.g., Co(II)) at 10-100 uM maximum in bacterial systems. Even that is tricky, as we need some trace metals to populate other metalloenzymes without introducing too much in the way of zinc-containing impurities.

Most zinc-metalloenzymes will be immune to metal chelation by DTT or BME, as the protein-metal binding constants will be orders of magnitude higher. (Values > 10^(12) are typical.) Even EDTA is not enough for many (most?) Cys(2)His(2) or Cys(2)His(OH2) sites. It is very unlikely that 1-5 mM DTT will be able to extract Zn from a metalloenzyme binding site. (We stored a particularly unstable Cys-rich zinc-metalloprotein in 100 mM DTT(!) and 2 mM EDTA (!!) and 50% glycerol and it is stable indefinitely at -20 deg C without detectable Zn loss.)

You may have to evaluate the metal-binding strength of your protein experimentally or by comparison to homologous proteins. If the binding constan is expected to be >10^(10), I don't think you need to worry too much about DTT or BME.


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 8/15/2014 11:03 AM, Harvey Rodriguez wrote:
Dear all,

Sorry for the non-crystallographic question. Currently I am working on a zinc binding protein which is expressed in insect cells and may contain 4-6 zinc ions. As we know, so many zinc binding proteins can absorb the iron ions from the culture medium and the protein looks from yellow to dark red when concentrated. But when I concentrate the protein, I didn’t see the red color even in the very high concentration. I am just wondering if a zinc binding protein is expressed from insect or mammalian cells, can the zinc binding sites grab the irons instead of zinc or the zinc binding site can be empty loaded if there is not enough zinc in the culture medium? If so, do I need to include some zinc salt into the culture medium when doing expression or I can add some zinc ions when purifying? Usually, how much zinc and at which step of purification can we add the zinc into the solution when doing purification?

Another question is that we know DTT can react with the heavy atoms to form the insoluble sulfide precipitates and if the zinc binding protein is purified with DTT at a final concentration of 1-5 mM, can it strip the zinc ions from the protein?

I am appreciated if someone has this kind of experimental experiences and thanks in advance!


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