Hi, I've seen soluble aggregates many times, with MBP and without. It's one of the common outcomes of partially successful (sounds better than saying 'failed') expression attempts.
It is always possible to push further and try some fairly esoteric stuff with E. coli if you're truly desperate, like induction at 4C in the presence of alcohol, or perhaps induction under salt shock in the presence of osmolytes (betaine), DMSO, etc. However, to me it appears that you may have reached a point where expression in a higher-order system might be required. Insect cell expression, yeast, or mammalian are all within reach of pretty much any decent lab these days - also there are numerous external companies and service providers who do that kind of work for relatively small amounts of money. This may be one of many cases where it's cheaper to try a different expression system than to beat one's head (or one's student/postdoc/staff scientist/good friend) against the wall to make E. coli work. Artem On Sat, Nov 13, 2010 at 4:04 PM, Jerry McCully < [email protected]> wrote: > Dear ALL; > > A few weeks ago, I posted my problem with MBP fusion protein. > > Thank folks very much for the help. Here is my latest result. > 1) Shortening the linker region would signifcantly reduce the cleavage of > target proteins from MBP; > > 2) Adding a C-terminal tag would minimize the degradation of target > proteins and facilitate the purification; > > 3) Low temperature induction(18 degree) would significantly reduce the > soluble aggregates; > > However, all the optimization procedures resulted in a ~150KD-200KD > oligomer of MBP fusion protein after gel-filtration. > Only a very small portion of MBP fusion protein exsited as monomers even in > the expression condition with low temperature and 0.1mM IPTG. > > Does anyone experience the oligomerization of MBP fusion protein? > > Thanks again and have a nice weekend! > > Jerry McCully > > > > > > > > >
