>Ive been expressing GFP fusions in e.coli and have been seeing bright
>subcellular localization. I'm concern that these may be inclusion
>bodies. Does anyone have any experience looking at GFP fusions in coli?
>Has anyone observed fluorescent inclusions bodies? I'd appreciate
>hearing from anyone with observations in this area. Thanks.
>>Robert E. Hughes
>University of Washington
my understanding is that GFP expressed in inclusion bodies (if as fusion or
alone) is non-fluorescent. When I initially expressed GFP from a T7
promoter in BL21 cells I got very high levels of GFP, but all of it was
expressed as insoluble, non-fluorescent inclusion bodies. Changing culture
conditions (temperature, media etc.) did not help at all. In the end I used
a different (weaker) promoter (lac promoter from Bluescript vector) and got
sufficient levels of functional, soluable protein (at 37 degree Celsius).
Hope that helps you,