Overexpressed E.Coli Protein sticks DNA - help needed!
sm-postmaster at ic.ac.uk
Fri Mar 8 04:14:01 EST 1996
I am trying to purify a his-tagged fusion protein in E.coli. It expresses
well, although comes out in inclusion bodies. After solubilisation in 6M
urea we can dilute and dialyse away the denaturant without precipitating
the protein - so far so good...!
Anyway we tried to put the protein over a s-sepharose column and hardly
anything would stick. We put a sample in the spec and did a 250nm-350nm
scan and saw lots of DNA. So we thought adding DNase/RNase would help -
added the two enzymes and finally the magnesium (MgCl2) and the whole lot
precipitated.We tried the components on their own and it is the magnesium
which is precipitating the mixture - we checked the pellet and it is all
My questions are - why does the magnesium precipitate the proteins (even
at 1mM), how can we avoid having the DNA in our prep, is there anything
we can try with the resulting pellet?
By the way, we have tried taking the pH down to about 5 and this
precipitates the protein as well.
Thanks in advance for any help / suggestions.
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