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A general approach for stabilizing nanobodies for intracellular expression

Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intrac...

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Detalles Bibliográficos
Autores principales: Dingus, John G, Tang, Jonathan CY, Amamoto, Ryoji, Wallick, Grace K, Cepko, Constance L
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9683787/
https://www.ncbi.nlm.nih.gov/pubmed/36416528
http://dx.doi.org/10.7554/eLife.68253
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author Dingus, John G
Tang, Jonathan CY
Amamoto, Ryoji
Wallick, Grace K
Cepko, Constance L
author_facet Dingus, John G
Tang, Jonathan CY
Amamoto, Ryoji
Wallick, Grace K
Cepko, Constance L
author_sort Dingus, John G
collection PubMed
description Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli. These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.
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spelling pubmed-96837872022-11-24 A general approach for stabilizing nanobodies for intracellular expression Dingus, John G Tang, Jonathan CY Amamoto, Ryoji Wallick, Grace K Cepko, Constance L eLife Cell Biology Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli. These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention. eLife Sciences Publications, Ltd 2022-11-23 /pmc/articles/PMC9683787/ /pubmed/36416528 http://dx.doi.org/10.7554/eLife.68253 Text en © 2022, Dingus et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Cell Biology
Dingus, John G
Tang, Jonathan CY
Amamoto, Ryoji
Wallick, Grace K
Cepko, Constance L
A general approach for stabilizing nanobodies for intracellular expression
title A general approach for stabilizing nanobodies for intracellular expression
title_full A general approach for stabilizing nanobodies for intracellular expression
title_fullStr A general approach for stabilizing nanobodies for intracellular expression
title_full_unstemmed A general approach for stabilizing nanobodies for intracellular expression
title_short A general approach for stabilizing nanobodies for intracellular expression
title_sort general approach for stabilizing nanobodies for intracellular expression
topic Cell Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9683787/
https://www.ncbi.nlm.nih.gov/pubmed/36416528
http://dx.doi.org/10.7554/eLife.68253
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