title: Usefulness of a Darwinian system in a biotechnological application: evolution of optical window fluorescent protein variants under selective pressure.
abstract: With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.
authors.affiliation: Department of Radiation Sciences - Research Unit Radiation Cytogenetics, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schoetz
authors.affiliation: Institute for Biological and Medical Imaging, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Nikolaos C
authors.initials: NC
authors.lastname: Deliolanis
authors.affiliation: Research Group Cellular Dynamics, Max Planck Institute of Neurobiology, Martinsried, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Ng
authors.affiliation: BAM Federal Institute for Materials Research and Testing, Division Biophotonics, Berlin, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Pauli
authors.affiliation: BAM Federal Institute for Materials Research and Testing, Division Biophotonics, Berlin, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Resch-Genger
authors.affiliation: Institute of Developmental Genetics, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Enrico
authors.initials: E
authors.lastname: Kühn
authors.affiliation: Department of Radiation Sciences - Research Unit Radiation Cytogenetics, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Heuer
authors.affiliation: Institute for Toxicology, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Beisker
authors.affiliation: Institute of Developmental Genetics, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Reinhard W
authors.initials: RW
authors.lastname: Köster
authors.affiliation: Department of Radiation Sciences - Research Unit Radiation Cytogenetics, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Zitzelsberger
authors.affiliation: Department of Radiation Sciences - Research Unit Radiation Cytogenetics, Helmholtz Center Munich - German Research Center for Environmental Health (GmbH), Munich, Germany.
authors.forename: Randolph B
authors.initials: RB
authors.lastname: Caldwell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001709:Biotechnology / Q000379:methods; D001717:Birds; D002460:Cell Line; D019020:Directed Molecular Evolution / Q000379:methods; D004625:Embryo, Nonmammalian; D005803:Genes, Immunoglobulin; D056084:Genetic Fitness / Q000502:physiology*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D061848:Optical Imaging / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid; D019076:Transgenes; D015027:Zebrafish
chemical_list: D008164:Luminescent Proteins