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:[EMAIL PROTECTED] On Behalf Of Kendall Nettles Sent: Friday, February 08, 2008 10:44 AM To: [email protected] 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
