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Re: [ccp4bb] Engineering disulfide bonds
 

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CCP4bb <-- 2008 <-- February 2008 <-- 08 February 2008
Previous message:
Subject: Re: Tag, you're in! Flag, V5, etc
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-02-08 		Next message:
Subject: Re: an over refined structure
From: Dale Tronrud det102 {- at -} UOXRAY {- dot -} UOREGON {- dot -} EDU
Date: 2008-02-08


Subject: Re: Engineering disulfide bonds
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-02-08
Hi Kendall,

As to (1) - it's a hard task. Redox potentials of Cys residues vary with
their environment, so it is very difficult to predict what might happen in
any specific case. Mixtures of reduced and oxidized GSH are commonly used to
maintain a specific redox environment, however if you're really unlucky -
some of your un-oxidized cysteins would be above and some below the system's
optimum redox potential. If your goal allows this - try protecting the
reduced Cys with thiol-reactive reagents, this won't affect the pre-formed
disulphide.

As to (2) - it's not easy to predict what happens - yes E. coli is a
reducing environment but I've seen several examples of SS bonds formed in
Coli-derived proteins. Presumably, these bonds formed during purification.

Journal of Molecular Biology Volume 315, Issue 1, 4 January 2002, Pages 1-8
- paper describing the use of FA113 strain of E. coli that is deficient in
both Trx and GSR, not sure if this is what the Origami cells are based on.

Happy expressionizing,

Artem

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Kendall Nettles
Sent: Friday, February 08, 2008 10:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Engineering disulfide bonds

I'm trying to engineer a disulfide bond into a protein that has several
other cysteines.

My question is whether there is a crystallization friendly reducing agent
that can be used to prevent oxidation of the free cysteines without breaking
the disulfide?

Also, can I expect 100% disulfide formation from standard bacterial
expression (assuming good geometry of the cysteines)?


Thanks,

Kendall
--
Kendall W. Nettles, PhD
Assistant Professor
Department of Cancer Biology
The Scripps Research Institute
5353 Parkside Dr.
Jupiter Fl 33458

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CCP4bb <-- 2008 <-- February 2008 <-- 08 February 2008
Previous message:
Subject: Re: Tag, you're in! Flag, V5, etc
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-02-08 		Next message:
Subject: Re: an over refined structure
From: Dale Tronrud det102 {- at -} UOXRAY {- dot -} UOREGON {- dot -} EDU
Date: 2008-02-08


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