As a comment; I've heard (somewhere) about people co-purifying
chaperones on Ni2+ -columns. The problems ware apparently solved by
adding ATP - probably releasing a protein-chaperone complex.
Kresten
In article <38398CA3.998DC9B0 at umh.es>,
=?iso-8859-1?Q?Jes=FAs?= Sanz <jmsanz at umh.es> wrote:
> Dear all,
>> I'm sorry if this is and old one, but I have just come across this
> problem.
> When trying to purify my protein from E. coli as a His-tag fusion, I
end
> up co-purifiying
> a very abundant 25 Kda protein (according to SDS-PAGE and mass
> spectrometry)
> that elutes at the same imidazole concentrations.
> My protein is found in inclusion bodies as well as in the soluble
> extract, but the
> contaminant is also found in the soluble and insoluble fractions.
>> I carried out an amino acid analysis and found plenty of histidines
and
> Asx + Glx.
> The database shows that an E. coli peptidyl-prolyl isomerase of the
> FKBP type has almost
> the same percent composition, (and these proteins have been found to
> bind to Ni2+
> columns very tightly) but its Mw is only around 21 Kda.
>> FYI, I'm using the pLEX system (Invitrogen) in the GI724 E. coli
strain.
>> If this subject is already in the FAQ, please let me know where can I
> access to it.
> Thank you very much
> ------------------------
> Prof. J. M. Sanz
> Centro de Biologia Molecular y Celular
> Universidad Miguel Hernandez
> E-mail: jmsanz at umh.es> WWW:http://cbmc.umh.es/jmsanz/jesussanz.htm> ----------------
>>
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Kresten Lindorff Larsen, Dept. Yeast Genetics
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