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Dear Ose, This is unfortunately a fairly common issue. There are multiple ways to rationalize what's happening - and even more approaches to reducing the aggregation issues. It would take too long to list all the options here - however I would look at the earliest stages of your expression and purification and try to determine if aggregation is the 'inevitable outcome' of inherent properties of your protein - or is it the outcome of inadequate expression, handling during lysis, purification, etc. Beyond the abovementioned considerations, the following may be useful to think about: a) is the specific activity of your protein close to maximum attainable (or at least biologically reasonable) value? b) if you can separate aggregates away from monomers (or proper oligomers) - does the protein re-aggregate with time or upon concentration? c) does aggregation state of your protein change with time or during the course of purification? d) what is the effect of ionic strength, pH, nature of buffer components, additives, ligands (substrates?), and so forth? (a) may be difficult to answer unless you have literature or unpublished data on this enzyme or its close relatives (b) is one of the earliest experiments I'd try in the absence of all other knowledge (c) should be tracked as you work with the protein as soon as you suspect that there may be issues (d) is probably the second experiment I'd try w/o any other knowledge. If you can use Thermofluor or some other high-throughput method to analyze the behavior of your protein exposed to various conditions, then you can screen quite an array of options very quickly Good luck, Artem "Nothing is built on stone; all is built on sand, but we must build as if the sand were stone" Jorge Luis Borges -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of ose toyoyuki Sent: Wednesday, August 26, 2009 7:55 PM To: [log in to unmask] Subject: [ccp4bb] Active aggregates? 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