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Dear Laurie, your cysteines might all be cytines, i.e. all paired and not really be the reason for the aggregation. Since you have soluble protein as long as MBP is tagged to it, why don't you test a large number of conditions for solubility? I'd probably start with a 3-D screen testing 2-4 different salts at different salt concentrations (e.g. 0-1M) and pH values in eppendorfs, cleave the MBP, spin it down and run S/N versus pellet on SDS-PAGE. That's easily done and might help. Cheers, Tim On Tue, Nov 16, 2010 at 11:13:18AM -0500, Laurie Betts wrote: > All - > > We are trying to express for structural studies a 257 AA eukaryotic > intracellular (also possibly nuclear) protein (predicted to be single domain > all-helical) that has 12 Cysteines. No known metal-binding function not > that it couldn't happen. So far (E. coli) it expressed solubly as MBP > fusion (with an N-terminal region deleted predicted disordered) until > cleavage of MBP, then it's not soluble, including detergents added. THe MBP > fusion is usually soluble aggregate so we assume that our part is not folded > right. We have so far assumed it needs a lot of reducing agent (5 mM DTT or > TCEP). Thinking of trying chaperones and insect cells next. > > Any experience out there that might help? Mostly I wonder about all the > cysteines. Don't really know if that is the problem. > > Laurie Betts -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A