Tandem phage-display for the identification of non-overlapping binding pairs of recombinant affinity reagents
| Autor: | Lauren C Roby, Kevin T. Gorman, Renhua Huang, Brian K. Kay, Allison M. Giuffre |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Dna template Analyte Phage display Enzyme-Linked Immunosorbent Assay Plasma protein binding Biology Polymerization law.invention 03 medical and health sciences 0302 clinical medicine Peptide Library law Genetics Humans Protein Interaction Domains and Motifs Peptide library Tandem Affinity Labels Combinatorial chemistry Recombinant Proteins High-Throughput Screening Assays 030104 developmental biology Genetic Techniques Reagent Recombinant DNA Methods Online Cell Surface Display Techniques Protein Binding 030215 immunology |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkx688 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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