Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies

Abstract 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 accur...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Yusei Matsuzaki, Wataru Aoki, Takumi Miyazaki, Shunsuke Aburaya, Yuta Ohtani, Kaho Kajiwara, Naoki Koike, Hiroyoshi Minakuchi, Natsuko Miura, Tetsuya Kadonosono, Mitsuyoshi Ueda
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6ca696081ad148fc8e2a41f6d690936f
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:6ca696081ad148fc8e2a41f6d690936f
record_format	dspace
spelling	oai:doaj.org-article:6ca696081ad148fc8e2a41f6d690936f2021-11-08T10:52:35ZPeptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies10.1038/s41598-021-01019-62045-2322https://doaj.org/article/6ca696081ad148fc8e2a41f6d690936f2021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01019-6https://doaj.org/toc/2045-2322Abstract 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.Yusei MatsuzakiWataru AokiTakumi MiyazakiShunsuke AburayaYuta OhtaniKaho KajiwaraNaoki KoikeHiroyoshi MinakuchiNatsuko MiuraTetsuya KadonosonoMitsuyoshi UedaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yusei Matsuzaki Wataru Aoki Takumi Miyazaki Shunsuke Aburaya Yuta Ohtani Kaho Kajiwara Naoki Koike Hiroyoshi Minakuchi Natsuko Miura Tetsuya Kadonosono Mitsuyoshi Ueda Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies
description	Abstract 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	article
author	Yusei Matsuzaki Wataru Aoki Takumi Miyazaki Shunsuke Aburaya Yuta Ohtani Kaho Kajiwara Naoki Koike Hiroyoshi Minakuchi Natsuko Miura Tetsuya Kadonosono Mitsuyoshi Ueda
author_facet	Yusei Matsuzaki Wataru Aoki Takumi Miyazaki Shunsuke Aburaya Yuta Ohtani Kaho Kajiwara Naoki Koike Hiroyoshi Minakuchi Natsuko Miura Tetsuya Kadonosono Mitsuyoshi Ueda
author_sort	Yusei Matsuzaki
title	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_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_sort	peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/6ca696081ad148fc8e2a41f6d690936f
work_keys_str_mv	AT yuseimatsuzaki peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT wataruaoki peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT takumimiyazaki peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT shunsukeaburaya peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT yutaohtani peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT kahokajiwara peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT naokikoike peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT hiroyoshiminakuchi peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT natsukomiura peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT tetsuyakadonosono peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies AT mitsuyoshiueda peptidebarcodingforonepotevaluationofsequencefunctionrelationshipsofnanobodies
_version_	1718442533104648192

Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies

Ejemplares similares