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 Zn(2+) or Cu(2+) 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.
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Genetics Institute, Inc., 87 CambridgePark Drive, Cambridge, MA 02140, USA. bct2@psu.edu
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Bourque
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Sookdeo
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McCoy
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D002845:Chromatography; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D015032:Zinc