I'm studying two proteins which are supposed to make a disulphide bridge between two cys. I have over-expressed the two proteins in vitro and I would like to induce disulphide formation. Do you have any suggestions on protocol to do that?
refolding at reducing conditions in presence of GSH enhances disulfide bond formation... i would suggest to go for strains that can form di sulphide bonds in vivo, as in vitro conditions has to be optimized and efficiency is an another question........
The issue here is the impairment of native disulfide-bonded form of the protein expressed and unfolding. Oxidation of cysteine thiols and the correction of mis-oxidized substrates have to be accomplished, and that would yield functionally active proteins. In a one-step procedure, the protein in question is diluted (100-fod) with buffer containing DTT. You may want to confer: Methods in Enzymology, volumes 107 and 131. If you are looking for a commercially available disulfide bond promoter New England Bio Labs would have one.
I have synthesized the disulfide di nucleosides recently. 5'-thiobenzoate nucleoside in saturated methanolic ammonia leads to the formation of disulfide bond eaasily.
In your case use basic conditions for disulfide formation check it outonce if your moity is stable in basic media
Chithan is correct if you are dealing with inclusion bodies. I use a strain of BL21 DE3 E. coli called Rosetta gami to overexpress globular proteins that contain disulfide bonds in order to avoid having to refold these types of protein. I have had very good luck using this method. The one thing I would caution you about if doing this is to make sure the temperature is not too high after inducing protein expression as this can result in the formation of inclusion bodies.
Rudolph, R., and Lange, C. (2008). Chapter 2-2 Strategies for the Oxidative in vitro Refolding of Disulfide-bridge-containing Proteins. In Oxidative Folding of Peptides and Proteins, L. Moroder, and J. Buchner, eds. (Cambridge: Royal Society of Chemistry), pp. 192–219.
Rudolph, R., and Lilie, H. (1996). In vitro folding of inclusion body proteins. FASEB J. 10, 49–56.
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