Hello people, Thank you very much for so many replies you've gone to for me including direct emails. This is a little summary of all the replies as a token of my appreciation. > 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. > By the way, can you observe a monomer peak during ultracentrifuge? In terms of > DLS, how about the polydispensity? HPV E6 also aggregates to similar size, but > the granules were relatively homogeneous. More importantly, it was observed > that in cell the protein could still form such large aggregates. Maybe for > some proteins, they were just meant to be like this. And based on my very > limited knowledge, most structures of such proteins remained as the > "higher-hanging fruit". > > Could you provide the reference of your success story? My protein also formed > large soluble aggregates and I am desperate for such successful stories! > >> 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. (In my case, I can't check if the specific activity of the recombinant protein is close to maximum attainable value or not, just I can say the activity of the aggregate is quite high. I can't separate aggregates away from monomers (or proper oligomers) because all of the newly prepared species are the aggregates. Perfect active aggregates! ) 1. First thing is, it's worth setting some trays even though it shows aggregation.(at least it’s the way not to waste your purified protein…)
2. Optimize the buffer condition: checking the effect of ionic strength, pH, nature of buffer components, additives, ligands (substrates), mild denaturant, and detergents. 3. Check the effects of those additives: DLS, Thermofluor, SAXS, and a transmission EM by negative stain. I wonder if Thermofluor works for this purpose, though (The aggregate might be more thermally stable than the monomer). > 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 regards toyoyuki -- Toyoyuki Ose o...@sci.hokudai.ac.jp Faculty of Advanced Life Science Hokkaido University N21W11 Kita-ku, Sapporo 001-0021 Japan
