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Dear James, Could you provide the reference of your success story? My protein also formed large soluble aggregates and I am desperate for such successful stories! By the way, have your performed DLS to your protein? What about the polydispensity? Is it lower than 20%? Thanks in advance! Sincerely, Xuan Yang 2009/8/27 James Stroud <[log in to unmask]> > Just try crystallizing it. What is a crystal but a "massive aggregate"? > That they are still soluble and active is great news. > > As a grad student, I had a similar phenomenon with an early project. I > showed a gel in group meeting where both activity and aggregation were > obvious, said the aggregate was no problem, got ridiculed when I said I was > going to throw it in trays despite what anyone said, had giant crystals > after a few trays, and solved the structure with miras. > > Get the structure and then worry about why it's aggregating. The structure > will probably provide you with the clues you need. > > > On Aug 26, 2009, at 5:55 PM, ose toyoyuki wrote: > > >> 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 >> [log in to unmask] >> >> Graduate School of Life Science >> Hokkaido University >> N21W11 Kita-ku, Sapporo >> 001-0021 Japan >> >