I'm working on building some FRET reporters. In addition to a cleavage site (of varying composition from 15-18AA), a 1 AA linker, I'm using Venus and Cerulean.
I am now working on further optimization. Does the removal of the intial methionine cause any problems for Venus if it is the second protein in the FRET pair? The basic layout is:
Cerulean - Link - Cleavage - Link - Venus
And I'm wondering if cutting the Met from Venus might bring me 1 AA closer and therefore a slightly better signal. I have seen it published both ways, and I was wondering if anyone had tried both or just knew theoretically that it wouldn't matter.