title: Generation of a dual-functional split-reporter protein for monitoring membrane fusion using self-associating split GFP.
abstract: Split reporter proteins capable of self-association and reactivation have applications in biomedical research, but designing these proteins, especially the selection of appropriate split points, has been somewhat arbitrary. We describe a new methodology to facilitate generating split proteins using split GFP as a self-association module. We first inserted the entire GFP module at one of several candidate split points in the protein of interest, and chose clones that retained the GFP signal and high activity relative to the original protein. Once such chimeric clones were identified, a final pair of split proteins was generated by splitting the GFP-inserted chimera within the GFP domain. Applying this strategy to Renilla reniformis luciferase, we identified a new split point that gave 10 times more activity than the previous split point. The process of membrane fusion was monitored with high sensitivity using a new pair of split reporter proteins. We also successfully identified new split points for HaloTag protein and firefly luciferase, generating pairs of self-associating split proteins that recovered the functions of both GFP and the original protein. This simple method of screening will facilitate the designing of split proteins that are capable of self-association through the split GFP domains.
authors.affiliation: Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.
authors.forename: Hirohito
authors.initials: H
authors.lastname: Ishikawa
authors.forename: Fanxia
authors.initials: F
authors.lastname: Meng
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Kondo
authors.forename: Aikichi
authors.initials: A
authors.lastname: Iwamoto
authors.forename: Zene
authors.initials: Z
authors.lastname: Matsuda
mesh_terms: D002459:Cell Fusion; D002460:Cell Line; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D049452:Green Fluorescent Proteins