Thanks to all for your help and suggestions. We are still working out the best protocol for our specific protein, but I list here the suggestions received, since they may prove helpful to others as well.
1. I assume that your native (12 Cys) protein runs as monomer on non-reducing SDS-PAGE and on native conditions, right? If your 13 Cys protein is still correctly folded, the dimerisation has happened spontaneously, but can be competed off by a proper redox system, e.g. oxidized and reduced glutathione: GS-SG, GSH; I would start with 1 mM GS-SG and 10 mM GSH, i.e. excess reduced GSH; the effect would be that disulfides tend to be reduced, unless they are conformationally protected. However, the other ratio might work as well: 10 mM GS-SG and 1 mM GSH. Now you would expect your Cys13 to be bonded to one of the excess glutathione (mixed disulfed), while the native disulfides remain intact, as they are conformationally stabilized. Of course, you can use a redox system other than glutathione, e.g. cysteine - cystine. Good luck, Hans 2. Did you inhibit S-S bond formation after addition of SDS? Lots of proteins form dimers when unfolded without presence of reducing agent. Adding 20 mM NEM into SDS-PAGE loading buffer is easiest way to prevent this. Assuming that you did this and you actually see intermolecular bond, you can play with reducing agent(s) concentrations. Intramolecular bonds require much higher concentration of reducing agents (e.g., IgG is perfectly happy and not reduced at 1 mM DTT at room temperature). Dima 3. For the first part (i.e. able to selectively reduce the protein at the engineered Cys without breaking the 6 disulfide bonds), this will depend on the solvent accessibility of these disulfide bonds. We have some examples of proteins containing up to 3 disulfide bonds and also free SH groups implicated in the catalysis for which we are able to add up to 2.5 mM of a reducing agent (DTT) without breaking S-S bonds. For the second part, you can use a reversible and specific thiol protecting group, such as MMTS. By doing this you form a S-methylthio-derivative of your protein, which can be easily reduced. Best regards, Rene Wintjens 4. Have you tried selective reduction using cysteine? Cysteine is used to reduce Fab2' to Fab' after pepsin digestions. It tends to reduce surface disulfides and leaves internal disulfides intact. However, you will need to experimentally determine the concentration of cysteine to use. I would try a concentration range of 0.5-20 mM cysteine. Hope this helps. Jeff 5. 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 Original query: > 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) >
