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Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies
Optimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate inves...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8563947/ https://www.ncbi.nlm.nih.gov/pubmed/34728738 http://dx.doi.org/10.1038/s41598-021-01019-6 |
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author | Matsuzaki, Yusei Aoki, Wataru Miyazaki, Takumi Aburaya, Shunsuke Ohtani, Yuta Kajiwara, Kaho Koike, Naoki Minakuchi, Hiroyoshi Miura, Natsuko Kadonosono, Tetsuya Ueda, Mitsuyoshi |
author_facet | Matsuzaki, Yusei Aoki, Wataru Miyazaki, Takumi Aburaya, Shunsuke Ohtani, Yuta Kajiwara, Kaho Koike, Naoki Minakuchi, Hiroyoshi Miura, Natsuko Kadonosono, Tetsuya Ueda, Mitsuyoshi |
author_sort | Matsuzaki, Yusei |
collection | PubMed |
description | Optimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence–function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody–antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in K(D) at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence–function relationships. |
format | Online Article Text |
id | pubmed-8563947 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-85639472021-11-04 Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies Matsuzaki, Yusei Aoki, Wataru Miyazaki, Takumi Aburaya, Shunsuke Ohtani, Yuta Kajiwara, Kaho Koike, Naoki Minakuchi, Hiroyoshi Miura, Natsuko Kadonosono, Tetsuya Ueda, Mitsuyoshi Sci Rep Article Optimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence–function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody–antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in K(D) at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence–function relationships. Nature Publishing Group UK 2021-11-02 /pmc/articles/PMC8563947/ /pubmed/34728738 http://dx.doi.org/10.1038/s41598-021-01019-6 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Matsuzaki, Yusei Aoki, Wataru Miyazaki, Takumi Aburaya, Shunsuke Ohtani, Yuta Kajiwara, Kaho Koike, Naoki Minakuchi, Hiroyoshi Miura, Natsuko Kadonosono, Tetsuya Ueda, Mitsuyoshi Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies |
title | Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies |
title_full | Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies |
title_fullStr | Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies |
title_full_unstemmed | Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies |
title_short | Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies |
title_sort | peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8563947/ https://www.ncbi.nlm.nih.gov/pubmed/34728738 http://dx.doi.org/10.1038/s41598-021-01019-6 |
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