Hi Heng, DTT can react with metal cations such as Zinc or Iron. This is why people to tend to use little DTT or no DTT at all on metal affinity columns or replace it b-mercaptoethanol or TCEP that do not interfere. Regarding the incorporation of Zinc into the culture media. I recall that Zinc finger domain expression in E coli (for nuclear receptors dna binding domains) is classically described to be done in presence of Zinc acetate added into the culture media (around 100 microMolar I think ) this is already a lot of zinc (more might be toxic to the cells).
depending on what your process of purification is. I would try to add zinc in the expression media (this has probably been descrived also for insect cells) if you use a Ni or Co chelating column I would NOT add any free zinc at this stage, you can, if you wish it, add it to eluate of your affinity column and to gel filtration buffers or ion exchange buffers. That said, I would suspect that having enough zinc around is mostly beneficial at the expression stage as the protein folding machinery is dealing with your target protein.zinc misloaded protein is likely to be unsoluble. for nuclear receptors DBDs there are protocols describing reincorporation (or even exchange) of metal ions inside the domain (maybe even starting from inclusion bodies) but the more cysteines you have the more likely it is to be difficult to get the "right" folded protein back. so I would rather favor a strategy trying to get as much folded protein as possible by natural means (let the cells do what we biochemists still don t know to do very well). if you add free zinc I would be careful with the concentration and the pH of your buffers. at basic pH zinc and other ions (such as Ca and Fe) form insoluble hydroxydes. if you have managed to purify some protein I would try to do some emission spectroscopy to see what ions are bound (zinc and or iron ) you may be surprised by what you will see. sorry for the lengthy response but I hope this helps. all the best, Pascal Egea On Fri, Aug 15, 2014 at 8:03 AM, Harvey Rodriguez < h.rodriguez.x...@gmail.com> 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! > > Heng > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
