Overexpressed E.Coli Protein sticks DNA - help needed!

non-authenticated sm-postmaster at ic.ac.uk
Fri Mar 8 04:14:01 EST 1996


Hi

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 
protein.

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.

Andy




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