title: Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro.
abstract: Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology. In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage. Target-specific variants with typically high nanomolar or low micromolar affinities (K(D)) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses. Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1. In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner. Possible intracellular applications of the selected affibody molecules are discussed.
authors.affiliation: Division of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Grimm
authors.forename: Emma
authors.initials: E
authors.lastname: Lundberg
authors.forename: Feifan
authors.initials: F
authors.lastname: Yu
authors.forename: Seiji
authors.initials: S
authors.lastname: Shibasaki
authors.forename: Erik
authors.initials: E
authors.lastname: Vernet
authors.forename: Marie
authors.initials: M
authors.lastname: Skogs
authors.forename: Per-Åke
authors.initials: PÅ
authors.lastname: Nygren
authors.forename: Torbjörn
authors.initials: T
authors.lastname: Gräslund
mesh_terms: D000595:Amino Acid Sequence; D000939:Epitopes / Q000378:metabolism; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000939:Epitopes; D011993:Recombinant Fusion Proteins; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins