Re: [ccp4bb] co-express two proteins in E.coli

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

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From: "Titia K {- dot -} Sixma" t {- dot -} sixma {- at -} NKI {- dot -} NL
Date: 2012-07-17

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Subject: Re: Large unit cell, overlaps
From: Jim Pflugrath Jim {- dot -} Pflugrath {- at -} RIGAKU {- dot -} COM
Date: 2012-07-17

Subject: Re: co-express two proteins in E.coli
From: Laurie Betts laurie {- dot -} betts0508 {- at -} GMAIL {- dot -} COM
Date: 2012-07-17

We have also had success expressing two proteins separately and then mixing
the soluble fractions of the lysates in the presence of a
complex-initiating ligand, where one protein has a His-tag (preferably the
protein that expresses to a lower extent) and the other no affinity tag
followed by a Nickel affinity column to capture the complex (unpublished).

Laurie Betts
UNC Chapel Hill

On Mon, Jul 16, 2012 at 11:55 PM, Biswajit Pal wrote:

> I fully agree with Dima. We are able to co-express and purify two
> interacting partners using pET28 and pET21 in E. coli. Some related
> references are :
>
> J. Mol. Biol. (2011) 405,49–64
> J. Biol. Chem. (2006) 281, 26491–26500
> J Struct Biol. (2011) 175(2):159-70
>
> Biswajit
>
>
> ----- Original Message -----
> From: "Dima Klenchin"
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Tuesday, July 17, 2012 5:16:34 AM
> Subject: Re: [ccp4bb] co-express two proteins in E.coli
>
> >I have been using the Duet system from Novagen (or whatever it is called
> >these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of
> >my proteins did not work in either vector. Either, one protein expressed
> >or the other. I played around with the promotors (they are both T7) by
> >changing one to the tac promotor. This increased the expression of this
> >gene but shut off expression of the other. The only way I could get my
> >proteins to co-express was to use pGEX vector with one protein, and
> >pRSFDuet with the other protein (leaving the second MCS empty). There is a
> >paper which sums up co-expression in E coli.
> >
> >http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-55.html
>
> There is really absolutely no problem co-expressing two proteins both from
> near-identical pET vectors as long as the two plasmids carry difference
> selection marker. We've used pET24 in combination with pET28 or pET31 on
> several complexes and it always works as long as you keep both antibiotics
> around.
>
> - Dima
>

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

Next message:
Subject: Re: Large unit cell, overlaps
From: Jim Pflugrath Jim {- dot -} Pflugrath {- at -} RIGAKU {- dot -} COM
Date: 2012-07-17