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TurboNuclease digests DNA and RNA to 1-4
base long fragments. It’s very useful to remove nucleic acids during protein
purification and virus purification. Another benefit of using TurboNuclease at
cell lysis is to significantly reduce lysate viscosity. So it reduces the
lysate volume for column loading. We can make a lysate of only 1L for 300 grams
of insect cells and load a Ni column without back pressure problem. We can use
lower g-force for lysate clearing. We got much lower 260 reading in the Ni pool
as well.
The specific activity of TurboNuclease (~1.3
units per ng) is thousands of fold higher than DNaseI. So the minute quantity
of TurboNuclease used has no interference with down stream applications.
--Chun
Competing Interests Statement
Accelagen is the maker of TurboNuclease.
From: CCP4 bulletin
board [mailto:[log in to unmask]] On
Behalf Of Pascal Egea
Sent: Monday, August 10, 2009 4:00
PM
To: [log in to unmask]
Subject: Re: [ccp4bb] DNA binding
protein
Hi Neeraj,
An absorption spectra between 220 and 400 nm (for example) should show
you if there is DNA coming along with your protein. In theory A280 is about 1.7
times A260 for a pure protein sample. This is a rough estimate. If your peak is
shifted towards 260 instead of 280 then you can suspect the presence of
contaminating DNA or RNA.
As to get rid of the DNA, I can suggest several ways to do it.
1-An heparin affinity column could out-compete your contaminating DNA
while binding your protein. They work really well.
2-Ion exchange is worth trying. It has worked for me with an extremely
basic archeal RNA binding protein purified in E.coli and bringing along some
endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating
nucleic acid, however with some loss of protein.
3-DNAse treatment may be worth trying but the presence of traces of
DNase may be a problem with subsequent crystallization trials in presence of
the bona-fide target DNA sequence.
4-Precipitation of the DNA with streptomycine sulfate. You could also
loss some protein.
1 and 2 are in my opinion the less invasive soutions.
Hope this helps.
Best regards
Pascal Egea