Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags

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

Previous message:
Subject: Re: low B factors
From: Pete Meyer pameyer {- at -} MCW {- dot -} EDU
Date: 2008-11-11

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Subject: Re: crystallization of proteins with His-tag and/or c-myc tags
From: Christopher Bahl ccp4 {- dot -} bahl {- at -} GMAIL {- dot -} COM
Date: 2008-11-11

Subject: Re: crystallization of proteins with His-tag and/or c-myc tags
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2008-11-11

On Nov 11, 2008, at 16:12, Tim Gruene wrote:

> Hi Jorge,
>
> using a system where you can cleave off the His-tag (with TEV,
> FactorX, etc) adds a purification step which is complementary to the
> first purification step (Ni-column etc). In my experience this
> results in very pure protein which makes it more likely to
> crystallise.

3C protease seems to work really well and would recommend it over TEV
or FX.
One could look at the His-SUMO tag ideas for alternative idea for
cleavage and some aid in solubilization.

> Therefore I would always choose such a system and not spend much
> time on trying to crystallise the protein with the His-tag attached.
>

If you have the His-protein always try to crystalize it. We got one
very nice structure with a His-zipper from the Tag helping to form the
lattice.
Consider the no-tag as yet another alternative for crystallization
trials - but if that does not work cleave it !

A.

> Tim
>
> --
> Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
> GPG Key ID = A46BEE1A
>
>
> On Tue, 11 Nov 2008, iulek@INTERPONTA.COM.BR wrote:
>
>> Dear all,
>>
>> Concerning the crystallization of proteins with a His-tag, based
>> upon discussions on this bulletin board and on the article
>>
>> Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette
>> and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst.
>> D63, 295-301, (2007).
>>
>> I understand that as a general approach one should try to
>> crystallize the protein with the His-tag (yet it might crystallize,
>> so
>> no need to care the work of taking out the tag); then, if this is not
>> successful, go to the procedure to either express (and purify) it
>> without the His tag or take it out later. Any observations/advices?
>> But one other question to add is what if the protein is expressed
>> with both a c-myc tag and a His-tag (you might consider also the
>> case
>> in which only the c-myc tag is present). Any experience on the
>> effect of
>> the c-myc tag on crystallization? References are welcome yet I
>> could not
>> find much googling around...
>> Thanks,
>>
>> Jorge

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

Previous message:
Subject: Re: low B factors
From: Pete Meyer pameyer {- at -} MCW {- dot -} EDU
Date: 2008-11-11

Next message:
Subject: Re: crystallization of proteins with His-tag and/or c-myc tags
From: Christopher Bahl ccp4 {- dot -} bahl {- at -} GMAIL {- dot -} COM
Date: 2008-11-11