Bc. Tripp et al., Investigation of the 'switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions, PROTEIN ENG, 14(5), 2001, pp. 367-377
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 bindin
g. 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 w
ere 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 chro
matography matrix, similar to a 'histidine-patch' thioredoxin variant, and
was reversibly precipitated by 1 mM Zn2+ or Cu2+ ions. Residues important f
or zinc and antibody binding were determined by site-directed mutagenesis.
The Trxloop antibody affinity was increased by saturation mutagenesis. Biot
inylated Trxloop ('Biotrxloop') variants of the original and improved affin
ity 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 wer
e dependent on the amount of immobilized Biotrxloop protein, indicating an
influence of avidity on the apparent affinity.