title: Splitting and self-assembling of far-red fluorescent protein with an engineered beta strand peptide: application for alpha-synuclein imaging in mammalian cells.
abstract: 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.
authors: BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon, Republic of Korea.
authors: Joo Oak
authors: JO
authors: Keem
authors: In Hwan
authors: IH
authors: Lee
authors: Sun Young
authors: SY
authors: Kim
authors: Yongwon
authors: Y
authors: Jung
authors: Bong Hyun
authors: BH
authors: Chung
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D051844:alpha-Synuclein / Q000032:analysis*; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D051844:alpha-Synuclein; D000097573:Red Fluorescent Protein