Facile isolation of high-affinity nanobodies from synthetic libraries using CDR-swapping mutagenesis

Abstract

Summary: The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies.For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021).

Keywords

• Cell Biology
• Flow Cytometry/Mass Cytometry
• Molecular Biology
• Antibody
• Protein Biochemistry
• Protein expression and purification