Hi,
I am trying to purify a 135 kDa protein, predicted pI=5.2. I can
efficiently express my protein as a fusion with MBP. Affinity
purification on amylose resin works fine at pH 6.2 while at pH 8.0 most
of the MBP fusion does not bind to the column. This has to do with
charge distribution/neutralization but it is (probably) irrelevant to my
problem. After elution from amylose resin, I cleave off MBP and the
protein I obtain is a large Mw aggregate. Purity determined after
SDS-PAGE is no more than 60%. I tried up to 10% glycerol, 1 M NaCl, 10
mM DTT and a number of detergents. I obtained some very slight
improvement with the above conditions and CHAPS as the detergent, but I
still have large aggregates as determined both by GPC and DLS.
Desperate, I decided to try and unfold/refold my protein, which is NOT
an enzyme and for which protein I do NOT have a functional assay! So far
I tried denaturing with 1%SDS, 8M urea, 6M guanidine. In the presence of
SDS I seem to obtain some reduction in the size of the aggregate (GPC,
DLS), while with urea and guandidine I do not see any change (DLS) in
the size of aggregate!
Any comment or suggestion? Is it possible that proteins aggregate
irreversibly?
Thanks,
Carlo
