Re: [ccp4bb] SUMMARY: Cannot run NTA to purify the protein having Histag?

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CCP4bb <-- 2007 <-- March 2007 <-- 05 March 2007

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From: Bart Hazes bart {- dot -} hazes {- at -} UALBERTA {- dot -} CA
Date: 2007-03-05

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Subject: Re: SUMMARY: Cannot run NTA to purify the protein having Histag?
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-03-05

Hi,
to add to your list:

You could also try Ni-IDA (e.g. from Pharmacia, I hope I remember the name
correctly) instead of Ni-NTA. If I remember correctly, IDA chelates the
metal ion only two-fld instead of three-fold as the NTA does.

During my PhD th protein would not bind at all to NTA but it work greatly
with IDA - and I mean 'not at all' while the protein expressed at 40-60
mg/l LB.

Cheers, Tim

--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

On Thu, 1 Mar 2007, Ngo Duc Tri wrote:

> Dear CCP4 users,
> Thank you all of your advices which help me to solve this problem. I believe
> that all of your advices will help me and others having the same problem.
> This is the summary of experienced advices about how you will do if the
> protein having His-tag don't bind to NTA resin.
>
> 1. The protein is aggregated or misfolded in such as way as to hide the His
> tag, try adding things like glycerol or detergents to try to prevent
> aggregation
> 2. Use mono-q or mono-s as a first step. After that, running the NTA resin.
> This way is helpful when dealing with insect cells.
> 3. Dialyze the lysate to remove contaminants that prevent binding. This is
> very common for Bv expression, but somewhat unusual for EC
> 4. Partial unfold of protein by including 1-3 M Urea in the buffer A
> 5. Incubate the protein with the resin in an end-over-end nutator to
> increase the interaction time or run the flow-through over a fresh batch of
> resin and do this several times until getting enough protein
>
> 6. Change the tag from e.g the N-terminus to the C-terminus ? Or if you have
> a structural homolog you could add the His tag into a loop, which is
> exposed.
> 7. Introduce a linker between the protein and the his-tag or create a 8xhis
> or 10xhis tag to enhance binding to the nta matrix
> 8. Using the Strep-II instead of His-tag
>
> My best regards,
> TriNgo
> Sungkyunkwan University
>

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CCP4bb <-- 2007 <-- March 2007 <-- 05 March 2007

Previous message:
Subject: Re: practical limits of MR?
From: Bart Hazes bart {- dot -} hazes {- at -} UALBERTA {- dot -} CA
Date: 2007-03-05

Next message:
Subject: Re: Problems in MR
From: Leo Chavas leo {- at -} PFWEIS {- dot -} KEK {- dot -} JP
Date: 2007-03-05