Selection and characterization of peptides binding with high affinity to metal ions using a 21‐mer random phage‐displayed peptide library

We have constructed a 21‐mer random phage‐displayed peptide library with a diversity of ~ 1x10 9 clones. Screening of this primary library against nickel (Ni‐NTA‐agarose) yielded peptides containing the HxH motif. A 13‐residue secondary library (diversity of >10 6 ) containing this motif was constructed and further screening with this library yielded a number of peptides with diverse sequences. The peptides were expressed as GST‐fusions in an E. coli system and quantitative data for the affinity to the metal ion was obtained using Surface Plasmon Resonance (BIACOR). Equilibrium dissociation constants ranged from the low micromolar for the weakest binding to the picomolar for the tightest binding peptides. FPLC experiments using NTA‐agarose and other metal ions indicated that the order of affinity of the peptides to metal ions was Ni > Cu > Zn. We have expressed concatamers of the highest affinity peptides in fusion with the chitin binding domain (CBD) and demonstrated binding specificity to nickel‐NTA agarose as well as chitin resin. Given the high levels of soluble protein expression, the relatively low cost of chitin and the high concentration of metal‐binding sites in the concatamer (picomolar dissociation constants and avidity), our system provides a feasible opportunity to remove metal ions from solution by a simple centrifugation or filtration process.