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. Crystals of many proteins have been obtained when the cells were lyzed with TurboNuclease, a few without using affinity purification. TurboNuclease is protease-free and endotoxin-free. --Chun Competing Interests Statement Accelagen is the maker of TurboNuclease. _____ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Pascal Egea Sent: Monday, August 10, 2009 4:00 PM To: CCP4BB@JISCMAIL.AC.UK 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
