Phage Display and Selection of Protein Ligands

Abstract

Introduction .................................................................................................................................... 101 Filamentous Phage Biology ........................................................................................................... 102M13 Virion.......................................................................................................................... 103 M13 Phage Life Cycle......................................................................................................... 105Phage Proteins Used for Display.................................................................................................... 106 Protein pIII ......................................................................................................................... 106 Protein pVIII ....................................................................................................................... 107 Nomenclature of Phage Display Experiments .................................................................... 107 Phagemid and Helper Phage ............................................................................................... 107Considerations for Successful Phage Display Experiment ............................................................ 107 Peptide Library ................................................................................................................... 108 Display of Proteins ............................................................................................................. 109 Antibody Libraries .............................................................................................................. 109Construction of the Phage Library ................................................................................................. 109 Library Diversity ................................................................................................................ 111Selection (Biopanning) Experiment............................................................................................... 111 Expression of Identied Protein or Synthesis of Peptide ............................................................... 113 Common Difculties ...................................................................................................................... 113 Conclusions .................................................................................................................................... 114 References ...................................................................................................................................... 114One of the principal modes of controlling biochemical processes in cells relies on protein binding to a ligand. The ligand could be another protein, a nucleic acid, or a small molecule. Phage display is a tool that makes possible the derivation of protein ligands that have never previously been known to exist in nature, and later, to use them in many types of experiments to study molecular interactions. The principle behind phage display is to make a protein or peptide in a bacterial cell, immobilize it on a phage particle, and tether to the peptide a molecular signature that fully describes the sequence of the peptide. This signature is a nucleic acid sequence that becomes part of the phage genome carried on the same phage particle as the displayed peptide and actually encodes the displayed protein. Different protein sequences can be displayed by simply modifying the sequence of the DNA through molecular engineering. The collective display of a large number of molecules on the phage is called a phage library.