Tandem phage-display for the identification of non-overlapping binding pairs of recombinant affinity reagents

• Published in: Nucleic acids research 2017-10, Vol.45 (18), p.e158-e158
• Main Authors: Gorman, Kevin T, Roby, Lauren C, Giuffre, Allison, Huang, Renhua, Kay, Brian K
• Format: Article
• Language: English
• Subjects: Affinity Labels - metabolism; Cell Surface Display Techniques - methods; Enzyme-Linked Immunosorbent Assay; Genetic Techniques; High-Throughput Screening Assays - methods; Humans; Methods Online; Peptide Library; Polymerization; Protein Binding; Protein Interaction Domains and Motifs; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkx688

The 'sandwich' binding format, which uses two reagents that can bind simultaneously to a given analyte, is the gold standard in diagnostics and many biochemical techniques. One of the bottlenecks in creating a sandwich assay is identifying pairs of reagents that bind non-competitively to the target. To bridge this gap, we invented Megaprimer Shuffling for Tandem Affinity Reagents (MegaSTAR) to identify non-competitive binding pairs of recombinant affinity reagents through phage-display. The key innovation in MegaSTAR is the construction of a tandem library, in which two reagents are randomly-displayed on the phage surface. This is accomplished by using a pool of 300-nucleotide long 'megaprimers', which code for previously-selected reagents, to prime second strand synthesis of a single-stranded DNA template and generate millions of pair-wise combinations. The tandem library is then affinity selected to isolate pairs that both reagents contribute to binding the target. As a proof-of-concept, we used MegaSTAR to identify pairs of fibronectin type III monobodies for three human proteins. For each target, we could identify between five and fifteen unique pairs and successfully used a single pair in a sandwich assay. MegaSTAR is a versatile tool for generating sandwich ELISA-grade and bispecific reagents.