Phage Display Panning on Silica: Optimization of Elution Conditions for Selection of Strong Binders

Phage display panning is a powerful tool to select strong peptide binders to a given target, and when applied to inorganic materials (e.g., silica) as a target, it provides information on binding events and molecular recognition at the peptide-mineral interface. The panning process has limitations with the phage chemical elution being affected by bias toward positively charged binders, resulting in the potential loss of information on binder diversity; the presence of fast growing phages with an intrinsic growth advantage; and the presence of false positives from target unrelated peptides. To overcome some of these limitations, we developed a panning approach based on the sequential use of different eluents (Gly-HCl, pH-2.2; MgCl₂, pH-6.1; and TEA, pH-11.0), or pH conditions (Gly-HCl 2.2 < pH < 11.0) that allows the identification of a diverse and comprehensive pool of strong binders. We have assessed and tested the authenticity of the identified silica binders via a complementary experimental (in vivo phage recovery rates and TEM imaging) and bioinformatics approach. We provide experimental evidence of the nonspecificity of the Gly-HCl eluent as typically used. Using a fluorimetric assay, we investigate in vitro binding of two peptides that differ by pI-S4 (HYIDFRW, pI 7.80) and S5 (YSLKQYQ, pI 9.44)─modified at the C terminal with an amide group to simulate net charges in the phage display system, confirming the vital role of electrostatic interactions as driving binding forces in the phage panning process. The presented optimized phage panning approach provides an opportunity to match known surface interactions at play with suitable elution conditions; to select only sequences relevant to a particular interfacial system. The approach has the potential to open up avenues to design interfacial systems to advance our understanding of peptide-assisted mineral growth, among other possibilities.