GFP protein not soluble? - (Aug/05/2005 )
I am purifying a His-GFP protein from BL21(DE3), the strange thing is .....
2 hr: cell pellet is not green
4 hr: light green
6 hr: green
Cell lysis result:
2hr: pellet and supernant are not green
4hr: pellet not green, supernant light green
6hr: pellet not green, supernant green
However, no matter at which time point and no matter what lysis method I use, e.g. sonication, bugbuster, freeze & thaw, I still found a large portion of the His-GFP protein remains in the lysed pellet. Does that means the GFP formed inclusion body? But the point is, if inclusion body is formed because of over expression, then how come after 2hr induction, the cell pellet is not green but I can find GFP in lysed pellet and no GFP released to the supernatant after cell lysis? Instead, it is found in the 6 hr supernatant which should have the highest expression and thus most inclusion body.
GFP is not fluorescent in its as-synthesized form. It requires an intra-molecular rearrangement of the amino acids to form a chromophore. Oxygen is a co-factor in this process. It takes time and oxygen to transform fully formed and folded GFP into fluorescent form.