Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Detalhes bibliográficos
Autor(a) principal: Gonçalves, Ana M.
Data de Publicação: 2022
Outros Autores: Pedro, Augusto Q., Oliveira, Diana M., Oliveira, Adriana E., Santos, Marino F. A., Correia, Márcia A. S., Queiroz, João A., Gallardo, Eugénia, Romão, Maria J., Passarinha, Luís A.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/145977
Resumo: Funding Information: This work was supported by Laboratório de Fármaco-Toxicologia, UBIMedical, and by Centro de Competências em Cloud Computing, co-financed by the European Regional Development Fund (ERDF) through the Programa Operacional Regional do Centro (Centro 2020), in the scope of the Sistema de Apoio à Investigação Científica e Tecnológica-Programas Integrados de IC&DT (Centro-01-0145-FEDER-000019-C4). This work was developed within the scope of the CICECO-Aveiro Institute of Materials (projects LA/P/0006/2020 ), and the Associate Laboratory Institute for Health and Bioeconomy—i4HB (project LA/P/0140/2020), which are financed by National Funds from FCT/MCTES. Augusto Q. Pedro research contract CEEC-IND/02599/2020 under the Scientific Stimulus—Individual Call from the Portuguese Foundation for Science and Technology (FCT) within the scope of POCH—Advanced Formation programs co-funded by European Social Fund and MCTES. Publisher Copyright: © 2022 by the authors.
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spelling Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferaseprotein activityprotein stabilitysoluble catechol-O-methyltransferase (SCOMT)thermal shift assay (TSA)CatalysisMolecular BiologySpectroscopyComputer Science ApplicationsPhysical and Theoretical ChemistryOrganic ChemistryInorganic ChemistrySDG 3 - Good Health and Well-beingFunding Information: This work was supported by Laboratório de Fármaco-Toxicologia, UBIMedical, and by Centro de Competências em Cloud Computing, co-financed by the European Regional Development Fund (ERDF) through the Programa Operacional Regional do Centro (Centro 2020), in the scope of the Sistema de Apoio à Investigação Científica e Tecnológica-Programas Integrados de IC&DT (Centro-01-0145-FEDER-000019-C4). This work was developed within the scope of the CICECO-Aveiro Institute of Materials (projects LA/P/0006/2020 ), and the Associate Laboratory Institute for Health and Bioeconomy—i4HB (project LA/P/0140/2020), which are financed by National Funds from FCT/MCTES. Augusto Q. Pedro research contract CEEC-IND/02599/2020 under the Scientific Stimulus—Individual Call from the Portuguese Foundation for Science and Technology (FCT) within the scope of POCH—Advanced Formation programs co-funded by European Social Fund and MCTES. Publisher Copyright: © 2022 by the authors.Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson’s disease. Currently, Parkinson’s disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography—IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and −80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.UCIBIO - Applied Molecular Biosciences UnitDQ - Departamento de QuímicaRUNGonçalves, Ana M.Pedro, Augusto Q.Oliveira, Diana M.Oliveira, Adriana E.Santos, Marino F. A.Correia, Márcia A. S.Queiroz, João A.Gallardo, EugéniaRomão, Maria J.Passarinha, Luís A.2022-12-02T22:16:27Z2022-102022-10-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article17application/pdfhttp://hdl.handle.net/10362/145977eng1661-6596PURE: 47766032https://doi.org/10.3390/ijms232012298info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:26:53Zoai:run.unl.pt:10362/145977Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:52:23.226404Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
title Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
spellingShingle Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
Gonçalves, Ana M.
protein activity
protein stability
soluble catechol-O-methyltransferase (SCOMT)
thermal shift assay (TSA)
Catalysis
Molecular Biology
Spectroscopy
Computer Science Applications
Physical and Theoretical Chemistry
Organic Chemistry
Inorganic Chemistry
SDG 3 - Good Health and Well-being
title_short Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
title_full Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
title_fullStr Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
title_full_unstemmed Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
title_sort Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
author Gonçalves, Ana M.
author_facet Gonçalves, Ana M.
Pedro, Augusto Q.
Oliveira, Diana M.
Oliveira, Adriana E.
Santos, Marino F. A.
Correia, Márcia A. S.
Queiroz, João A.
Gallardo, Eugénia
Romão, Maria J.
Passarinha, Luís A.
author_role author
author2 Pedro, Augusto Q.
Oliveira, Diana M.
Oliveira, Adriana E.
Santos, Marino F. A.
Correia, Márcia A. S.
Queiroz, João A.
Gallardo, Eugénia
Romão, Maria J.
Passarinha, Luís A.
author2_role author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv UCIBIO - Applied Molecular Biosciences Unit
DQ - Departamento de Química
RUN
dc.contributor.author.fl_str_mv Gonçalves, Ana M.
Pedro, Augusto Q.
Oliveira, Diana M.
Oliveira, Adriana E.
Santos, Marino F. A.
Correia, Márcia A. S.
Queiroz, João A.
Gallardo, Eugénia
Romão, Maria J.
Passarinha, Luís A.
dc.subject.por.fl_str_mv protein activity
protein stability
soluble catechol-O-methyltransferase (SCOMT)
thermal shift assay (TSA)
Catalysis
Molecular Biology
Spectroscopy
Computer Science Applications
Physical and Theoretical Chemistry
Organic Chemistry
Inorganic Chemistry
SDG 3 - Good Health and Well-being
topic protein activity
protein stability
soluble catechol-O-methyltransferase (SCOMT)
thermal shift assay (TSA)
Catalysis
Molecular Biology
Spectroscopy
Computer Science Applications
Physical and Theoretical Chemistry
Organic Chemistry
Inorganic Chemistry
SDG 3 - Good Health and Well-being
description Funding Information: This work was supported by Laboratório de Fármaco-Toxicologia, UBIMedical, and by Centro de Competências em Cloud Computing, co-financed by the European Regional Development Fund (ERDF) through the Programa Operacional Regional do Centro (Centro 2020), in the scope of the Sistema de Apoio à Investigação Científica e Tecnológica-Programas Integrados de IC&DT (Centro-01-0145-FEDER-000019-C4). This work was developed within the scope of the CICECO-Aveiro Institute of Materials (projects LA/P/0006/2020 ), and the Associate Laboratory Institute for Health and Bioeconomy—i4HB (project LA/P/0140/2020), which are financed by National Funds from FCT/MCTES. Augusto Q. Pedro research contract CEEC-IND/02599/2020 under the Scientific Stimulus—Individual Call from the Portuguese Foundation for Science and Technology (FCT) within the scope of POCH—Advanced Formation programs co-funded by European Social Fund and MCTES. Publisher Copyright: © 2022 by the authors.
publishDate 2022
dc.date.none.fl_str_mv 2022-12-02T22:16:27Z
2022-10
2022-10-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10362/145977
url http://hdl.handle.net/10362/145977
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 1661-6596
PURE: 47766032
https://doi.org/10.3390/ijms232012298
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 17
application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
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instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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