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Removal of contaminanting DNA from DNA binding protein inclusion body

Removal of contaminanting DNA from DNA binding protein inclusion body - Protein Forum

Removal of contaminanting DNA from DNA binding protein inclusion body - Protein Forum


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Old 07-21-2011, 01:25 AM
Pipette Filler
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Default Removal of contaminanting DNA from DNA binding protein inclusion body



This is a tough if not impossible one for anyone willing to give it a shot...
Purification of an insoluble DNA binding protein (~40kDa) in inclusion body via denaturization in 8M Urea.
Current protocol is
1. Lysis
2. 2M Urea Pellet wash + 50U Benzonase
3. 8M Urea + 1% Triton solubilization
4. IMAC

So by PICO Green analysis and agarose gel the Benzonase does the job in 2M Urea. Then when 8M Urea comes in I get the re-appearance of high bp fragments on the gel. This kind of makes some sense since the protein is within an inclusion body and is a DNA binding protein.

Here's the tough part. How can I shred up the residual DNA upon denaturization??
To make things even tougher I can not use any type of precipitation if you were thinking PEI as I was. Current purification is at our contract manufacturer and cant have any major changes.
Also, forget about re-folding prior to IMAC, that's a done deal too.

Benzonase in 7M Urea has a life of about 15 minutes so I think in 8M Urea it would be futile.

Any ideas? Add a ton of Benzonase at the solubilization step and hope for the best? I'm even doing an experiment now with 500U at the 2M Urea wash stage but it seems I'm getting the same thing (running the gel tomorrow) - just seems that way so far by PICO Green analysis.

I know this is crazy but worth asking.
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binding , body , contaminanting , dna , inclusion , protein , removal


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