[ccp4bb] Zinc binding protein expressed from insect cells
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
[ccp4bb] protein lost activity after size exclusion chromatography
Dear all, Recently, I came across an obstacle on the purification and acitivty measurement of my protein. My protein was expressed with an C terminal His tag in the HEK 293T cells and purified by nickel affinity, anion exchange and size exclucion chromatography. For every purification step, I preserved some sample to test the activty. Strikingly, the protein retains activity after nickel affinity column even for three days but lost almost all the activty immediately after Mono Q and SEC. Therefore, I speculated that something (metal ion or co-factor) binding to the protein was striped by the Mono Q column. Then I skipped this step and only use the SEC for further purification. However, the protein is still not active no matter what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel column is also in the PBS buffer and no additive was added. Buffer exchange in the concentrator doesn't affect the activity of the protein. Can anyone explain why anion exchange or size exclucion chromatography destroy the activity of the protein? Any comment or proposal is appreciated! Harvey
