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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2022
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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. |
format | Online Article Text |
id | pubmed-9683787 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
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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