Direct selection of EGF mutants displayed on filamentous phage using cellsoverexpressing EGF receptor

Understanding receptor-ligand interactions, and the signal transduction pat hways they activate, is of great interest for the discovery of novel antago nists and agonists. In this report we describe a rapid and efficient procedure to evaluate the importance of several different epidermal growth factor (EGF) residues for the binding and activation of its receptor (EGFR). We constructed an EGF mu tant library randomized at positions 13, 15 and 16 and expressed them on fi lamentous phages. Phage display is a powerful system, allowing rapid isolat ion of binding mutants. Since many of the most pharmacologically interestin g receptors cannot be produced in a soluble form, we developed a technique to rapidly select receptor-binding molecules directly on cells. A luciferas e assay, simple to perform, was then used to test their biological transduc tion activity and to rapidly detect mutants of interest. Analysis of the re sulting sequences revealed that the wild-type amino acids at positions 13, 15 and 16 are optimized for binding and activity. EGF mutants with agonist properties were also isolated and tolerated substitutions were identified.