Phage-Display Methodology for the Study of Protein-Protein Interactions: Stage 1, Generating Libraries

Abstract

INTRODUCTIONThis 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. Specifically, we used this protocol to generate libraries of domains in which we randomized six to eight codons that define the natural VH3 specificity of SpA. In our effort, these codons were variegated using the NNK or VNS doping strategy. Toward this goal, three large (60- to 88-residue) oligonucleotides with targeted degenerate codons were designed. To facilitate construction of the composite domain library, these oligonucleotides had overlapping complementary stretches of at least 12 nucleotides. Of course, for application to other domains, the number and composition of the required oligonucleotides are dependent on the specific variant domains to be generated. This protocol details Stage 1 of the method, the generation of a phage library via overlap PCR and bacterial cloning. The initial PCR amplification requires the mixing of equimolar amounts of two oligonucleotides to form a template for the subsequent PCR.