My recombinant protein appeared as triplet in SDS-PAGE!

Yasuyuki Fujimoto fujimoto at MOL.F.U-TOKYO.AC.JP
Mon Oct 26 04:08:20 EST 1998


Hi, all.

I am in trouble. My recombinant protein (purified from E. coli)
appeared as triplet bands of about 45, 47, 49 kDa in SDS-PAGE. I want
to know  how this happens and how I can avoid this. Immediately after
purification, the protein mainly appeares as single band of 45kDa.
Then, after over night at room temperature (my recombinant protein is
unstable and aggregates at 4 degree), the band of 47 and 49 kDa
appears. Most of the band of the 45 kDa gradually disappears within two
or three days. After the change, the  band pattern continues more than
three weeks at room temperature. However, incubation longer than one
week cause gradual appearance of bands of about 90, 140 kDa and other
larger ones suggesting formation of dimer, trimer and further oligomer
of the 45kDa protein. Total amount of bands does not seem to decrease.
In all these cases, protein concentration was 1 to 2 mg/ml. This
phenomenon was detected in solution of about 100 mM imidazole (pH 8.0)
or 100mM EDTA (purification was done using Nickel column).


 I think this phenomenon was not caused by protein
degradation(proteolysis) but by intra and inter molecular crosslinking
by some unknown mechanism.


Any suggestions will be appreciated. You can e-mail me directly at 

fujimoto at mol.f.u-tokyo.ac.jp


Thank you!





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