Some more details-problems with cleaving fusion protein
toudjarska at wi.mit.edu
Thu Apr 11 09:35:41 EST 2002
Thank you for your reply. Before considering changing the linker and
protease I was thinking of few more experiments to see if the problem isn't
elsewhere. I appreciate if you can give me some advise on the following.
When I purify the fusion with FPLC- monoQ something strange is happening.
After SDS-PAGE of all fractions it appears that my protein is eluting at 2
different salt concentrations- at around 200 mM and 500 mM NaCl. Does that
mean that some aggregates or complexes with other proteins are being
formed??? I have done protease cleavage for removing MBP only on protein
after amylose column before MonoQ. Now I am doing cleavage on protein
fraction 200 mM NaCl from MonoQ. And I will run gel filtration. BUT my
question is since I have done cleavage in presence of Tween 20 and Triton-X
100 (0.01 and 0.1%) isn't that going to disrupt any aggregation or
interaction with other proteins??? And if that (agregation or interaction)
is the problem cloning another protease clavage site probably won't help.
btw I need the protein for X-ray crystallography.
Thanks a lot for any input
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