Dear all, I am purifying a fusion protein with maltose-binding protein(MBP) as the N-term tag. As a control, I also purified free-MBP from empty vector. The vector I used is pMAL-c5x (from NEB) with an addition of precision protease cutting site (8 residues, LEVLFQGP) between MBP and multi-cloning site.
Then I run both protein through Superose6(size-exclusion chromatography) to check oligomerization states. To my surprise, both protein has two peaks, one is at the monomer size and another one has a big MW. SDS-gel shows from both protein, the small peaks are both free-MBP monomer, but the big peaks are fusion protein and free-MBP respectively. The estimated MW of the big peaks are similar to 10 folds of their corresponding monomers but heterogeneous (checked by DLS and native gel). To my knowledge, MBP should behave as a monomer. Does anyone have seen the similar thing before or could give some explanations? I suspect maybe the precision protease cutting site and the following multi-clonging site at the C-term of free-MBP might cause oligomerization, could it be possible? I am trying to delete this part to see whether it will improve, is there any other thing I can try in order to eliminate heterogeneity? Any suggestion or comment will be really appreciated. Thank you! Best regards, Xiao Xiao
