Splitting and self-assembling of far-red fluorescent protein with an engineered beta strand peptide: Application for alpha-synuclein imaging in mammalian cells

We introduce the strategic development of self-assembling peptide/protein fragments based on the far-red fluorescent protein mPlum. The first beta strand (mPlum 1, 18 amino acids) of mPlum was engineered to spontaneously bind with the rest of the protein (mPlum 2-11, next 10 beta strands) and to form a native chromophore. The target beta strand mPlum 1 was separated from mPlum 2-11 and linked via a flexible peptide linker, resulting in fluorescently inactive circularly permuted mPlum protein (CpmPlum). In vitro evolution of this CpmPlum to a fluorescently active form and the subsequent splitting of the engineered mPlum 1 peptide afforded self-assembling mPlum fragments. Recombinantly expressed and synthetically prepared beta strand peptides were successfully assembled with the remaining mPlum protein in vitro and in cells. This developed pair of peptide/protein fragments was effectively used for peptide tag detection of alpha-synuclein in mammalian cells. Sequential expression of self-assembling mPlum fragments offered an entirely genetically encoded sensing system of naturally unfolded alpha-synuclein.