Re: [ccp4bb] Removal of bacterial chaperone

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Removal of bacterial Hsp70 contaminant from recombinant protein
From: Brad Bennett saint_genevieve {- at -} ATT {- dot -} NET
Date: 2007-03-09

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From: Flip Hoedemaeker flip {- at -} KEYDP {- dot -} COM
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Subject: Re: Removal of bacterial chaperone
From: Martin Hallberg Martin {- dot -} Hallberg {- at -} KI {- dot -} SE
Date: 2007-03-09

Hi Yong,

Perhaps try one of these:

1. Wash with ATP+Mg when bound on the lMAC column (to get it to
release your protein).
2. Wash with 0.1% Triton when bound on the IMAC column.
3. Hydrophobic interaction chromatography.
4. Wash with 0.5 GuHCl while bound on the IMAC column (pray!!)

On a sad note, the protein will quite often aggregate when it is
released. There is a reason why the only sign of target protein you
see is stuck in a chaperone... but at least you have the chance to
scout around for a good buffer to release it into.

Martin

On Mar 9, 2007, at 6:39 PM, Yong Tang wrote:

> RE: Removal of bacterial chaperone Hsp70 contaminant from recombinant
> protein preparation
>
> Dear all,
>
> I have a protein expressed at 37C for 3 hours in BL21 DE3 and purified
> with sub-stoichiometric amount of apparent Hsp70 contaminant even
> after exhaustive affinity (GST-fusion or His-tagged), ion-exchange and
> sizing column steps. I would like to know if you have a
> well-established protocol for getting rid of such a contaminant. I
> asked around and was told to try adding ATP at certain(?) stage of the
> purification.
>
> I would much appreciate your input.
>
> Many thanks! –yong @ the Wistar Institute

.
B. Martin Hallberg, PhD
Molecular Cell Biology Program
Department of Cell and Molecular Biology
Karolinska Institutet
SE-171 77 Stockholm
Sweden

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Removal of bacterial Hsp70 contaminant from recombinant protein
From: Brad Bennett saint_genevieve {- at -} ATT {- dot -} NET
Date: 2007-03-09

Next message:
Subject: Re: wine coolers for crystallization?
From: Flip Hoedemaeker flip {- at -} KEYDP {- dot -} COM
Date: 2007-03-09