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Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification

Green fluorescent proteins (GFPs) are widely used in biological research. Although GFP can be visualized easily, its precise manipulation through binding partners is still burdensome because of the limited availability of high-affinity binding partners and related structural information. Here, we re...

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Autores principales: Zhang, Ziyue, Wang, Yao, Ding, Yu, Hattori, Motoyuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148334/
https://www.ncbi.nlm.nih.gov/pubmed/32277083
http://dx.doi.org/10.1038/s41598-020-62606-7
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author Zhang, Ziyue
Wang, Yao
Ding, Yu
Hattori, Motoyuki
author_facet Zhang, Ziyue
Wang, Yao
Ding, Yu
Hattori, Motoyuki
author_sort Zhang, Ziyue
collection PubMed
description Green fluorescent proteins (GFPs) are widely used in biological research. Although GFP can be visualized easily, its precise manipulation through binding partners is still burdensome because of the limited availability of high-affinity binding partners and related structural information. Here, we report the crystal structure of GFPuv in complex with the anti-GFP nanobody LaG16 at 1.67 Å resolution, revealing the details of the binding between GFPuv and LaG16. The LaG16 binding site was on the opposite side of the GFP β-barrel from the binding site of the GFP-enhancer, another anti-GFP nanobody, indicating that the GFP-enhancer and LaG16 can bind to GFP together. Thus, we further designed 3 linkers of different lengths to fuse LaG16 and GFP-enhancer together, and the GFP binding of the three constructs was further tested by ITC. The construct with the (GGGGS)(4) linker had the highest affinity with a K(D) of 0.5 nM. The GFP-enhancer-(GGGGS)(4)-LaG16 chimeric nanobody was further covalently linked to NHS-activated agarose and then used in the purification of a GFP-tagged membrane protein, GFP-tagged zebrafish P2X4, resulting in higher yield than purification with the GFP-enhancer nanobody alone. This work provides a proof of concept for the design of ultra-high-affinity binders of target proteins through dimerized nanobody chimaeras, and this strategy may also be applied to link interesting target protein nanobodies without overlapping binding surfaces.
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spelling pubmed-71483342020-04-15 Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification Zhang, Ziyue Wang, Yao Ding, Yu Hattori, Motoyuki Sci Rep Article Green fluorescent proteins (GFPs) are widely used in biological research. Although GFP can be visualized easily, its precise manipulation through binding partners is still burdensome because of the limited availability of high-affinity binding partners and related structural information. Here, we report the crystal structure of GFPuv in complex with the anti-GFP nanobody LaG16 at 1.67 Å resolution, revealing the details of the binding between GFPuv and LaG16. The LaG16 binding site was on the opposite side of the GFP β-barrel from the binding site of the GFP-enhancer, another anti-GFP nanobody, indicating that the GFP-enhancer and LaG16 can bind to GFP together. Thus, we further designed 3 linkers of different lengths to fuse LaG16 and GFP-enhancer together, and the GFP binding of the three constructs was further tested by ITC. The construct with the (GGGGS)(4) linker had the highest affinity with a K(D) of 0.5 nM. The GFP-enhancer-(GGGGS)(4)-LaG16 chimeric nanobody was further covalently linked to NHS-activated agarose and then used in the purification of a GFP-tagged membrane protein, GFP-tagged zebrafish P2X4, resulting in higher yield than purification with the GFP-enhancer nanobody alone. This work provides a proof of concept for the design of ultra-high-affinity binders of target proteins through dimerized nanobody chimaeras, and this strategy may also be applied to link interesting target protein nanobodies without overlapping binding surfaces. Nature Publishing Group UK 2020-04-10 /pmc/articles/PMC7148334/ /pubmed/32277083 http://dx.doi.org/10.1038/s41598-020-62606-7 Text en © The Author(s) 2020 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Zhang, Ziyue
Wang, Yao
Ding, Yu
Hattori, Motoyuki
Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification
title Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification
title_full Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification
title_fullStr Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification
title_full_unstemmed Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification
title_short Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification
title_sort structure-based engineering of anti-gfp nanobody tandems as ultra-high-affinity reagents for purification
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148334/
https://www.ncbi.nlm.nih.gov/pubmed/32277083
http://dx.doi.org/10.1038/s41598-020-62606-7
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