Journal
STAR PROTOCOLS
Volume 3, Issue 1, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.xpro.2021.101101
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Funding
- National Institutes of Health [RF1AG059723, R35GM136300]
- National Science Foundation [CBET 1159943, 1605266, 1813963, 1804313]
- Biointerfaces Institute
- Albert M. Mattocks Chair
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This study presents a simple method to isolate high-affinity nanobodies from common framework libraries using CDR-swapping mutagenesis. By shuffling the CDRs of low-affinity variants during the sorting process, high-affinity nanobodies can be directly isolated without the need for lead isolation and optimization. This approach, demonstrated here for SARS-CoV-2 neutralizing nanobodies, is expected to simplify the generation of high-affinity nanobodies.
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).
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