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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