Dear all, This is a question on how to cope with the protein that seems to form massive aggregates in solution but enzymatically active. I'm working on a protein whose molecular weight is around 70kDa and can be divided into two domains (say A and B domains). We expressed this protein in E.coli fused with GST and purified using some chromatography. The GST affinity chromatography works well and proteinase digestion to remove the tag does wonders too. The purified protein was confirmed to be active enough, we can detect both activities from these two domains. But the retention time from the gel filtration clearly shows it is awfully aggregated (comes out at the void region). DLS measurement indicates the averaged diameter is around 45 nm, which I feel is a bit too long. Analytical ultracentrifuge result implies that the distribution of the molecular species is wide, some portion got precipitated with 1K rcf (means the molecular weight is more than 5MDa) and the rest is ranging from 1MD to 5MDa with a peak at 1MDa. I made new two constructs covering the A and B domains respectively, both of them are active again, but only the A domain has got the same symptom as the intact protein. The B domain seems to exist as a monomer in solution. Here come my questions, (I) How can I interpret this phenomenon? (II) Is there anything we can try to change the situation? (III) Does it make sense to try crystallization? (probably not).(IV) Has anyone got such experience? I tried the methylation on lysine side chains, I also tried the buffer with 0.2M arginine or 10% glycerol but the all the results just seem hopeless. The protein before the proteinase treatment also comes out at the void region from the gel filtration. cheers
toyoyuki --? Toyoyuki Ose [email protected] Graduate School of Life Science Hokkaido University N21W11 Kita-ku, Sapporo 001-0021 Japan
