Direct Selection of EGF Mutants Displayed on Filamentous Phage Using Cells Overexpressing EGF Receptor
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C. Souriau
Abstract
Understanding receptor-ligand interactions, and the signal transduction pathways they activate, is of great interest for the discovery of novel antagonists 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 mutant library randomized at positions 13, 15 and 16 and expressed them on filamentous phages. Phage display is a powerful system, allowing rapid isolation of binding mutants. Since many of the most pharmacologically interesting receptors cannot be produced in a soluble form, we developed a technique to rapidly select receptor-binding molecules directly on cells. A luciferase assay, simple to perform, was then used to test their biological transduction activity and to rapidly detect mutants of interest. Analysis of the resulting 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.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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Artikel in diesem Heft
- Protein Kinase C-α Produces Reciprocal Effects on the Phorbol Ester Stimulated Tyrosine Phosphorylation of a 50 kDa Kinase in Jurkat Cells
- Growth- and Development-Dependent Expression of Gangliosides in Rat Hepatocytes and Liver Tissues
- Regulated Phosphorylation of the Gal4p Inhibitor Gal80p of <I>Kluyveromyces lactis</I> Revealed by Mutational Analysis
- The WW Domain of Dystrophin Requires EF-Hands Region to Interact with β-Dystroglycan
- Purification and MALDI-MS Characterization of Stressin, a Stress-Associated Glycoprotein
- Direct Selection of EGF Mutants Displayed on Filamentous Phage Using Cells Overexpressing EGF Receptor
- Refolding Studies on the Tetrameric Loop Deletion Mutant RM6 of ROP Protein
- Secretion of Protease Nexin-II/Amyloid β Protein Precursor by Human Colorectal Carcinoma Cells and Its Modulation by Cytokines/Growth Factors and Proteinase Inhibitors
- Isolation and Primary Structure of the CCI Papain-Like Cysteine Proteinases from the Latex of Carica candamarcensis Hook
- Identification of SLPI (Secretory Leukocyte Protease Inhibitor) in Human Mast Cells Using Immunohistochemistry and In Situ Hybridisation
- Stereochemistry of the Interaction between Methionine Sulfur and the Protein Core
- Trypsin from Pacifastacus leninsculus Hepatopancreas: Purification and cDNA Cloning of the Synthesized Zymogen
