Hi Artem,
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.
What will?
btw I need the protein for X-ray crystallography.
Thanks a lot for any input
Iva
