Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro.
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.
Grimm
Sebastian
S
Division of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Lundberg
Emma
E
Yu
Feifan
F
Shibasaki
Seiji
S
Vernet
Erik
E
Skogs
Marie
M
Nygren
Per-Åke
PÅ
Gräslund
Torbjörn
T
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*
D000939:Epitopes; D011993:Recombinant Fusion Proteins; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins