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 (KD) 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.