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Re: [ccp4bb] Engineering disulfide bonds
 

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CCP4bb <-- 2008 <-- February 2008 <-- 08 February 2008
Previous message:
Subject: Re: Engineering disulfide bonds
From: "David J {- dot -} Schuller" djs63 {- at -} CORNELL {- dot -} EDU
Date: 2008-02-08 		Next message:
Subject: Re: Engineering disulfide bonds
From: Kornelius Zeth kornelius {- dot -} zeth {- at -} TUEBINGEN {- dot -} MPG {- dot -} DE
Date: 2008-02-08


Subject: Re: Engineering disulfide bonds
From: Cynthia Kinsland clk10 {- at -} CORNELL {- dot -} EDU
Date: 2008-02-08
There are strains designed to provide a less-reducing (more
oxidizing) environment (Origami or any of it is "gami" derivatives
from Novagen).

They are deficient in the thioredoxin and glutathione reductases (I
think I recall...I didn't look it back up).

We've used them with good success for some disulfide requiring
proteins. However, they grow slowly and can be sickly-seeming and
annoying to work with.

You also have the option of sending (or, attempting to send) your
protein to the periplasm, which is a more oxidizing environment. The
pelB leader is available in a variety of pET vectors. IBA markets
several flavors of plasmid with the ompA leader sequence. There are
versions of the MBP that still contain its leader sequence, and the
DsbC and DsbA fusions (available on pET vectors) are designed for
periplasmic export.

We've made our own version of a pET plasmid with the OmpA leader
sequence and have used it to export toxic proteins (including a
nuclease) to the periplasm. Generally, I've found the OmpA leader to
be better than the pelB leader at getting proteins to the periplasm.

Best of luck,

Cynthia

On Feb 8, 2008, at 11:02 AM, David J. Schuller wrote:

> On Fri, 2008-02-08 at 10:44 -0500, Kendall Nettles wrote:
>
>> Also, can I expect 100% disulfide formation from standard bacterial
>> expression (assuming good geometry of the cysteines)?
>
> No, E. coli cells are a reducing environment.
>
> -
> ======================================================================
> =
> With the single exception of Cornell, there is not a college in the
> United States where truth has ever been a welcome guest - R.G.
> Ingersoll
> ======================================================================
> =
> David J. Schuller
> modern man in a post-modern world
> MacCHESS, Cornell University
> schuller@cornell.edu

____________________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844




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CCP4bb <-- 2008 <-- February 2008 <-- 08 February 2008
Previous message:
Subject: Re: Engineering disulfide bonds
From: "David J {- dot -} Schuller" djs63 {- at -} CORNELL {- dot -} EDU
Date: 2008-02-08 		Next message:
Subject: Re: Engineering disulfide bonds
From: Kornelius Zeth kornelius {- dot -} zeth {- at -} TUEBINGEN {- dot -} MPG {- dot -} DE
Date: 2008-02-08


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