Investigation of the `switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions

The `FLITRX' random peptide library, consisting of dodecamer loop peptides displayed on a thioredoxin-flagellin scaffold on Escherichia coli, was used to select peptide sequences with affinity for a monoclonal antibody. These peptides were further screened for pH- and metal-sensitive antibody binding. Several zinc-sensitive peptides were identified, termed `switch epitopes'. A soluble, monomeric thioredoxin loop (`Trxloop') insertion analog of a FLITRX switch epitope was constructed and its antibody binding properties were characterized by Western blots. Zinc-dependent antibody recognition was maintained in the Trxloop protein although the apparent antibody affinity was lower. This Trxloop protein bound to an immobilized metal affinity chromatography matrix, similar to a `histidine-patch' thioredoxin variant, and was reversibly precipitated by 1 mM Zn2+ or Cu2+ ions. Residues important for zinc and antibody binding were determined by site-directed mutagenesis. The Trxloop antibody affinity was increased by saturation mutagenesis. Biotinylated Trxloop (`Biotrxloop') variants of the original and improved affinity Trxloop proteins were constructed and characterized by surface plasmon resonance measurements. Increased antibody affinity was partially due to a slower antibody desorption rate, although the relative adsorption rates were dependent on the amount of immobilized Biotrxloop protein, indicating an influence of avidity on the apparent affinity.