3.3. Panning of MOrPH-PhD Library

This section outlines the procedure for panning the MOrPH-PhD library against an immobilized target protein of interest (biopanning). In the biopanning process, the library of phage-displayed MOrPHs is incubated with the immobilized target and the unbound phages are washed away. The protein-bound phages are eluted by lowering the pH and then amplified in E. coli. The resulting library is subjected to additional rounds of affinity selection and amplification (typically, 3–4 rounds in total) to enrich the pool with cyclic peptide sequences with high affinity toward the target protein. After the selection process, the individual clones are identified via DNA sequence and further characterized. In the protocol below, the MOrPH-PhD library from section 3.2 is panned against streptavidin immobilized on magnetic beads. For other target proteins, the protein of interest can be immobilized onto streptavidin- or neutravidin-coated (magnetic) beads via a biotin tag. Alternatively, the target protein can be immobilized on microtiter plate [50,53] or on other resins through non-specific adsorption, covalent capture, or through other affinity tags. After immobilization of the target protein, the capturing matrix (i.e. beads or plate) is blocked with bovine serum albumin (BSA) to minimize nonspecific binding of the phages to the matrix.