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Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9

The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine...

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Autores principales: Singh, Advaita Acarya, Pillay, Priyen, Naicker, Previn, Alexandre, Kabamba, Malatji, Kanyane, Mach, Lukas, Steinkellner, Herta, Vorster, Juan, Chikwamba, Rachel, Tsekoa, Tsepo L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9433777/
https://www.ncbi.nlm.nih.gov/pubmed/36061808
http://dx.doi.org/10.3389/fpls.2022.953654
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author Singh, Advaita Acarya
Pillay, Priyen
Naicker, Previn
Alexandre, Kabamba
Malatji, Kanyane
Mach, Lukas
Steinkellner, Herta
Vorster, Juan
Chikwamba, Rachel
Tsekoa, Tsepo L.
author_facet Singh, Advaita Acarya
Pillay, Priyen
Naicker, Previn
Alexandre, Kabamba
Malatji, Kanyane
Mach, Lukas
Steinkellner, Herta
Vorster, Juan
Chikwamba, Rachel
Tsekoa, Tsepo L.
author_sort Singh, Advaita Acarya
collection PubMed
description The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.
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spelling pubmed-94337772022-09-02 Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9 Singh, Advaita Acarya Pillay, Priyen Naicker, Previn Alexandre, Kabamba Malatji, Kanyane Mach, Lukas Steinkellner, Herta Vorster, Juan Chikwamba, Rachel Tsekoa, Tsepo L. Front Plant Sci Plant Science The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant ΔXTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and ΔXTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors. Frontiers Media S.A. 2022-08-18 /pmc/articles/PMC9433777/ /pubmed/36061808 http://dx.doi.org/10.3389/fpls.2022.953654 Text en Copyright © 2022 Singh, Pillay, Naicker, Alexandre, Malatji, Mach, Steinkellner, Vorster, Chikwamba and Tsekoa. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Singh, Advaita Acarya
Pillay, Priyen
Naicker, Previn
Alexandre, Kabamba
Malatji, Kanyane
Mach, Lukas
Steinkellner, Herta
Vorster, Juan
Chikwamba, Rachel
Tsekoa, Tsepo L.
Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
title Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
title_full Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
title_fullStr Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
title_full_unstemmed Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
title_short Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9
title_sort transient proteolysis reduction of nicotiana benthamiana-produced cap256 broadly neutralizing antibodies using crispr/cas9
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9433777/
https://www.ncbi.nlm.nih.gov/pubmed/36061808
http://dx.doi.org/10.3389/fpls.2022.953654
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