Hi, Your idea does sound really crazy but actually Jacob had made a very good valid point. Question is do you think your aggregate still functioning or not, if not, can you revive them in vitro and how effective is your refolding process if you are going to refold them?
You may want to take a look at structures of some bacterial pore-forming proteins. Many of them can be expressed as large inclusion bodies in heterologous host of which we call "aggregate". I know it sound fishy but after refolding and proper proteolytic activation, those proteins retain the ability to induce mortality to their host cell as good as the real one. Anyway, good luck with your endeavors! Puey On Tue, Feb 21, 2012 at 3:26 PM, Jacob Keller < j-kell...@fsm.northwestern.edu> wrote: > Hey, it could be that you just have a big oligomer--any support for > that in the relevant literature? A 10-mer would probably beat out an > s200, no? Do you have any other way to ascertain the oligomeric state? > > Jacob > > On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam > <r...@brandeis.edu> wrote: > > Hi Folks, > > > > As crazy as it sounds, if you have crystallized and managed to solve the > > structure of a protein from aggregated protein, please could you share > your > > experience. > > > > After many constructs, many many expression schemes and after the usual > > rigmarole of optimization that is also often discussed on ccp4bb > (buffers, > > glycerol, salt concentrations, pH, detergent, additives etc.), I now > have a > > decently expressing truncated construct for my protein (80 kDa) that is > pure > > but aggregated (elutes in the void volume from a Superdex200 column). I > am > > tempted to make a boatload of aggregated protein and set up some crystal > > trays (after perhaps testing by CD). So I'd like to hear from folks who > have > > been successful in solving structures from aggregates when many many > known > > and tested optimization methods still leave one with aggregated protein. > > > > Thanks. > > Raji > > > > -- > > Raji Edayathumangalam > > Instructor in Neurology, Harvard Medical School > > Research Associate, Brigham and Women's Hospital > > Visiting Research Scholar, Brandeis University > > > > > > > > -- > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > ******************************************* >
