Re: [ccp4bb] Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase

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CCP4bb <-- 2007 <-- November 2007 <-- 02 November 2007

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Subject: Re: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Roger Rowlett rrowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2007-11-02

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Subject: Re: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2007-11-02

Subject: Re: Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase
From: Raji Edayathumangalam redayath {- at -} MAIL {- dot -} ROCKEFELLER {- dot -} EDU
Date: 2007-11-02

How about trying two opposite ion-exchange columns??!! Namely, monitoring co-elution behaviour on
anion exchange and cation exchange. For example, we have a case where the contaminant and protein of
interest will both bind and co-elute from monoQ but only protein of interest or contaminant (can't
remember which) bound the monoS. This then led to separation of the two species....

If all else simple trials fail, how about using a cleavable His-tag on your protein, cleaving with
the specfic protease, rebinding on Ni-affinity and collecting your protein as the unbound fraction?
Can't tell from your description if this has been tried...

Not sure if Cobalt-chelated column makes a difference with respect to elution properties.

Raji

---------Included Message----------
>Date: 2-nov-2007 14:43:02 -0400
>From: "Ailong Ke"
>To:
>Subject: [ccp4bb] Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate
aminotransferase
>
>>Hello,
>
>
>We are trying to purify an N-terminal His6-tagged protein from E.
>coli, and the prep was contaminated with the E coli
>Glucosamine-fructose-6-phosphate aminotransferase, which co-purifies
>with my protein of interest in subsequent ion-exchange and sizing
>columns. This protein appears to be a common contamination in the
>Ni-NTA purifications. Does anyone have tricks to get rid of it?
>Thanks.
>
>Ailong
>
>
>
>--
>
>
---------End of Included Message----------

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