> 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
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