title: Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications.
abstract: Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolution, resulting in superior probes for use in live-cell fluorescence microscopy. In this protocol, we provide methods for engineering a monomeric FP, for enhancing its brightness by directed evolution, and for thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features, such as a red-shifted emission spectrum or enhanced photostability. Although the length of the procedure is dependent on the degree of optimization desired, the basic steps can be accomplished in 4-6 weeks.
authors.affiliation: Department of Chemistry, University of California, Riverside, California, USA.
authors.forename: Hui-Wang
authors.initials: HW
authors.lastname: Ai
authors.affiliation: National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Baird
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shen
authors.affiliation: National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D019020:Directed Molecular Evolution; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D019151:Peptide Library; D049452:Green Fluorescent Proteins