His-tag intruder

Lars Komorowski larskomo at biochem.mu-luebeck.de
Wed Dec 1 04:54:17 EST 1999


In fact it is the isomerase SLYD. It binds Ni. I have had the same problem
and we sequenced the N-terminus of the contaminant.
Lars

Jesús Sanz <jmsanz at umh.es> schrieb in im Newsbeitrag:
38398CA3.998DC9B0 at umh.es...
> 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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