Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro

✍ Scribed by Sebastian Grimm; Emma Lundberg; Feifan Yu; Seiji Shibasaki; Erik Vernet; Marie Skogs; Per-Åke Nygren; Torbjörn Gräslund

• Book ID: 103851118
• Publisher: Elsevier
• Year: 2010
• Tongue: English
• Weight: 488 KB
• Volume: 27
• Category: Article
• ISSN: 1871-6784
• DOI: 10.1016/j.nbt.2010.07.016

✦ Synopsis

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.