[RASMB] Common contamination in recombinant protein preparation

[Chad Brautigam]

Hello Dror and All,

Back in my graduate lab, we used several means to rid our preps of  
contaminating DNA.  I think Lizz points out a very important one:   
anion exchange chromatography.  We often used DEAE for this purpose.

Another strategy is differential precipitation.  We used Polymin P to  
precipitate the offending nucleic acid.  It's also conceivable that  
Ammonium Sulfate cuts could help.  However, if the protein of interest  
natively binds nucleic acid, co-precipitation could occur.

DNase treatment followed by SEC might also help.

Dror may have tried all of these, however-- it's unclear from his  
message.

Peace out,
Chad

On Jun 1, 2010, at 9:15 AM, Bartlett, Lizz wrote:

> Hi All,
> In industry DNA, etc is removed during the purification steps often  
> using an ion exchange method.
>
> Cheers!
> Lizz Bartlett
>
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org 
> ] On Behalf Of Hayes, David
> Sent: Tuesday, June 01, 2010 9:26 AM
> To: rasmb at rasmb.bbri.org
> Subject: Re: [RASMB] Common contamination in recombinant protein  
> preparation
>
> Hi Dror Noy,
>
> I am the AUC guy here at MedImmune, so I am not an expert:
> But colleagues here use intercalating dyes like pico green to measure
> DNA concentrations.  This may be complete overkill (especially the  
> metal
> enhanced version my colleagues developed) but you sound like you  
> really
> want to be certain if this is DNA and how much is there.  I include a
> reference to a recent paper.  But I also believe that there is a kit  
> and
> even a special mini-fluorometer called Quant-It that measures DNA,  
> RNA,
> and protein.  The kits were not quite good enough for our purposes  
> here,
> but may be sufficiently precise for your needs.
>
> I am too far downstream to know how they get rid of the extra DNA when
> they find it.
>
> 1: Dragan AI, Bishop ES, Casas-Finet JR, Strouse RJ, Schenerman MA,
> Geddes CD.
> Metal-enhanced PicoGreen fluorescence: application for double-stranded
> DNA
> quantification. Anal Biochem. 2010 Jan 1;396(1):8-12. Epub 2009 Sep  
> 11.
> PubMed
> PMID: 19748479.
>
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org 
> ]
> On Behalf Of Dror Noy
> Sent: Monday, May 31, 2010 2:52 AM
> To: rasmb at server1.bbri.org
> Subject: [RASMB] Common contamination in recombinant protein  
> preparation
>
> Not exactly AUC related but there's a chance to find here a few
> people who work with proteins and do care about their UV absorption
> properties:
> We make a variety of native and artificial recombinant proteins and
> often encounter a situation where the apparently pure fraction
> according to SDS-PAGE gels has its UV absorption peak blue shifted
> from the expected ~280 nm  toward 260 nm. This seems to be a general
> problem and usually happens when proteins are purified from inclusion
> bodies. We suspect DNA contamination but so far we were unable to get
> rid of it. I'd be very grateful for tips from people who experienced
> this phenomenon and were able to solve it.
> Thanks,
> Dror Noy
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