Protocol

Phage-Display Methodology for the Study of Protein-Protein Interactions: Stage 2, Panning

This protocol was adapted from "Phage-Display Methodology for the Study of Protein-Protein Interactions," Chapter 9, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 2 of the method, we describe the use of streptavidin-coated magnetic beads for library selection (panning). After selection, the elution strategy used to recover desirable clones should be chosen on the basis of the type of panning strategy employed, and the properties of the ligand that has been targeted. Of the several alternatives, we have compared the common recovery strategies of low pH, trypsinization of bound libraries, and competition with soluble antigen. When considering which option to employ, it is worth knowing how this may affect the type of binder that will be recovered. Low pH will dissociate most or all binders, whether they are specific or nonspecific for the selection antigen, whereas trypsin targets the fusion area between the protein and gene III, and specifically cleaves it. As a result, for the latter, only phage that bind via a protein-protein interaction will be recovered. In contrast, soluble antigen will provide specific binders of relatively high affinity because the elution will work in a competitive manner. We recommend an empiric approach to determine which is best for specific projects.