[RASMB] Common contamination in recombinant protein preparation

Ewa Folta-Stogniew ef55 at email.med.yale.edu
Tue Jun 1 10:26:24 PDT 2010


How would one identify presence and remove just a single nucleotide 
rather than nucleic acids?  I know one can see by 260 nm absorbance, 
but differentiate between polymer and a single nucleotide?

Ewa

At 12:51 PM 6/1/2010, Jeff Cohlberg wrote:
>I can second Lizz Bartlett's recommendation.  In purifying 
>recombinant superoxide dismutase from a yeast expression system, we 
>often found PicoGreen-positive DNA in protein that had been purified 
>with phenyl-Sepharose plus size exclusion, but little or none when a 
>DEAE-Sepharose ion exchange step was added.
>
>Jeff Cohlberg
>
>
>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: 
>><mailto:rasmb-bounces at rasmb.bbri.org>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: <mailto:rasmb at rasmb.bbri.org>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: 
>><mailto:rasmb-bounces at rasmb.bbri.org>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: <mailto:rasmb at server1.bbri.org>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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>
>
>--
>Jeffrey A. Cohlberg, Professor and Chair
>Department of Chemistry and Biochemistry
>California State University, Long Beach
>Long Beach, CA 90840
>562-985-4944            fax 775-248-1263
><mailto:cohlberg at csulb.edu>cohlberg at csulb.edu
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