Steven E. Cwirla

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage.

Large collections of random peptides can be expressed on the N-terminus of the pIII protein of filamentous phage and screened for binding to antibodies and other receptors. In our previous work with a monoclonal antibody (3E7) (Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382, 1990), we showed that a high proportion of the selected peptides had relatively low affinity (Kds greater than 1 microM). Here we describe conditions for selective enrichment of phage expressing high affinity peptides. This is done by allowing the phage to interact with a low concentration of 3E7 Fab followed by extensive washing to allow dissociation of phage-bearing peptides with low affinity. These affinity selection conditions were applied to the pool of phage previously selected using a high concentration of IgG. A phage clone with the known high affinity ligand YGGFL (Kd 7.1 nM) and several other closely related peptides were isolated. The dissociation rate of 125I-3E7 Fab from several phage clones approximated that of phage expressing YGGFL. A good correlation was found between the dissociation rate of the peptides found on phage and the equilibrium binding constants of chemically synthesized peptides. The strategy of using a low concentration of receptor and extensive washing to select phage-bearing high affinity peptides, combined with assays to determine the specificity and relative affinity of peptides on isolated phage clones, should be generally applicable in using the peptides-on-phage system for discovery of high affinity receptor ligands.