> We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro site-directed chemical modification. The mutant protein is well-expressed and soluble, but while we do see some monomer, non-reducing SDS-PAGE shows that a substantial proportion of it is probably in a homodimeric form (we suspect dimerization through intermolecular disulfide formation),
You could consider to reversibly protect the free cysteine chemically. Treat with sulfite + tetrathionate (ca 10-100 mM) to let the free cysteines react to S-sulfonates and prevent them forming disulfides. Later, when you want the free cysteines, you treet with thiols (ca. 1 mM). The problem of regioselectivity for the disulfide cleave, though, remains. Low DTT concentrations or redox buffers with glutathion (GSH/GSSG) could work. Gottfried =================================================================== WEB-Mailer der Uni-Greifswald ( http://www.uni-greifswald.de/ ) ===================================================================
