Cargando…
Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125
BACKGROUND: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9563788/ https://www.ncbi.nlm.nih.gov/pubmed/36242022 http://dx.doi.org/10.1186/s12934-022-01939-6 |
_version_ | 1784808487020134400 |
---|---|
author | Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa |
author_facet | Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa |
author_sort | Colarusso, Andrea |
collection | PubMed |
description | BACKGROUND: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues. RESULTS: Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology. CONCLUSIONS: Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural–functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce “intractable” proteins, especially those containing large intrinsically disordered regions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01939-6. |
format | Online Article Text |
id | pubmed-9563788 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-95637882022-10-15 Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa Microb Cell Fact Research BACKGROUND: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues. RESULTS: Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology. CONCLUSIONS: Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural–functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce “intractable” proteins, especially those containing large intrinsically disordered regions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01939-6. BioMed Central 2022-10-14 /pmc/articles/PMC9563788/ /pubmed/36242022 http://dx.doi.org/10.1186/s12934-022-01939-6 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 |
title | Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 |
title_full | Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 |
title_fullStr | Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 |
title_full_unstemmed | Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 |
title_short | Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 |
title_sort | active human full-length cdkl5 produced in the antarctic bacterium pseudoalteromonas haloplanktis tac125 |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9563788/ https://www.ncbi.nlm.nih.gov/pubmed/36242022 http://dx.doi.org/10.1186/s12934-022-01939-6 |
work_keys_str_mv | AT colarussoandrea activehumanfulllengthcdkl5producedintheantarcticbacteriumpseudoalteromonashaloplanktistac125 AT lauroconcetta activehumanfulllengthcdkl5producedintheantarcticbacteriumpseudoalteromonashaloplanktistac125 AT calvanesemarzia activehumanfulllengthcdkl5producedintheantarcticbacteriumpseudoalteromonashaloplanktistac125 AT parrilliermenegilda activehumanfulllengthcdkl5producedintheantarcticbacteriumpseudoalteromonashaloplanktistac125 AT tutinomarialuisa activehumanfulllengthcdkl5producedintheantarcticbacteriumpseudoalteromonashaloplanktistac125 |