Dear all, 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), and we also see other higher molecular weight species that are immunoreactive with an antibody to our protein, so maybe we have heterodimerization with other cysteine-containing proteins as well.
Can anyone point me to references or protocols that might help us to selectively reduce our protein at the engineered Cys, breaking up the dimers, while preserving the disulfide structure of the native protein? Or might there be a way to reversibly protect the engineered Cys throughout expression and purification to prevent dimerization? Any suggestions are welcome. Thanks! Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
