Overexpressed E.Coli Protein sticks DNA - help needed!
achim at ibex.ca
Tue Mar 19 15:59:42 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.
You do not give any conditions, like the buffer you used. In case you are using
phosphate buffer, the magnesium could precipitate it, especially if you also have
ammonium in your matrix. In case this is not the cause, you have to post some more
sample matrix, pH, buffer, Are you sure; chromatographic conditions. Are you sure
S-sepharose should work? Which protein is it?
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