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Re: [ccp4bb] Removal of bacterial Hsp70 contaminant from recombinant protein
 

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CCP4bb <-- 2007 <-- March 2007 <-- 09 March 2007
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Subject: ´ð¸´: Removal of bacterial Hsp7 0 contaminant from recombinant protein
From: "Wang, Yeming (NIH/NIEHS) [F]" WangY2 {- at -} NIEHS {- dot -} NIH {- dot -} GOV
Date: 2007-03-09 		Next message:
Subject: Re: Removal of bacterial chaperone
From: Martin Hallberg Martin {- dot -} Hallberg {- at -} KI {- dot -} SE
Date: 2007-03-09


Subject: Re: Removal of bacterial Hsp70 contaminant from recombinant protein
From: Brad Bennett saint_genevieve {- at -} ATT {- dot -} NET
Date: 2007-03-09
Hey Yong...
add 10 mM ATP + 2.5 mM MgCl2 in your purification buffer and wash it off during your affinity step...this has worked for me (of course make sure that you're not unlucky and your protein doesn't elute during this step; don't worry it shouldn't)
if that doesn't work, maybe try changing some things on the cell growth and expression end, like induction at lower temperature or a different BL21 strain like pLysS or STAR

HTH
Brad

--
Brad C Bennett
Postdoctoral Research Assistant
Department of Biochemistry,
Cellular and Molecular Biology
(BCMB)
University of Tennessee-Knoxville
865-974-3045
bennet02@utk.edu



-------------- Original message ----------------------
From: Yong Tang
> 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
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