ArticleInternational journal of molecular sciences2015
Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.
Article in International journal of molecular sciences, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- A Strategy for Simultaneous Engineering of Interspecies Cross-Reactivity, Thermostability, and Expression of a Bispecific 5T4 x CD3 DARTAntibodies (Basel, Switzerland) · 2025Article
- FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein.New biotechnology · 2021Article
- Carrier-Free CXCR4-Targeted Nanoplexes Designed for Polarizing Macrophages to Suppress Tumor Growth.Cellular and molecular bioengineering · 2019Article
- Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation.International journal of molecular sciences · 2019Review
- Ribosome Display: A Potent Display Technology used for Selecting and Evolving Specific Binders with Desired Properties.Molecular biotechnology · 2019Review
- Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement.Molecular pharmaceutics · 2018Article
- Generating FN3-Based Affinity Reagents Through Phage Display.Current protocols in chemical biology · 2018Article
- Tandem phage-display for the identification of non-overlapping binding pairs of recombinant affinity reagents.Nucleic acids research · 2017Article
- Generating Recombinant Antibodies to Membrane Proteins through Phage Display.Antibodies (Basel, Switzerland) · 2016Review
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Authors and funding
6 authors.
Funding
Abstract
Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold.
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