[ccp4bb] Zinc binding protein expressed from insect cells

2014-08-15 Thread Harvey Rodriguez
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!


[ccp4bb] protein lost activity after size exclusion chromatography

2011-03-16 Thread Harvey Rodriguez
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!