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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...

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Autores principales: Matsuzaki, Yusei, Aoki, Wataru, Miyazaki, Takumi, Aburaya, Shunsuke, Ohtani, Yuta, Kajiwara, Kaho, Koike, Naoki, Minakuchi, Hiroyoshi, Miura, Natsuko, Kadonosono, Tetsuya, Ueda, Mitsuyoshi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
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.
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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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