Overexpressed E.Coli Protein sticks DNA - help needed!

Marc Goldstein magoldst at ix.netcom.com
Fri Mar 15 22:04:21 EST 1996


Andy,

It is very possible that your DNA is stabilizing the protein. Is the
protein normally DNA-associated? You may find that it is not possible
to keep the protein in solution unless you give it some DNA to stick
to. If the large quantity of excess DNA is the problem, you may try
exchanging the E. coli genomic/plasmid DNA with shorter bits of DNA
produced by shearing, restriction, or synthesis. The protein may
migrate to the smaller strands if you incubate the mix together.

Marc


non-authenticated <sm-postmaster at ic.ac.uk> wrote:

>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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