Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34
Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the ye...
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| Dil: | İngilizce |
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2020
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| Online Erişim: | https://hdl.handle.net/20.500.12008/31779 |
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| _version_ | 1868889943224352768 |
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| author | Steglich Guarino, Martina |
| author2 | Lombide, Rodrigo López, Ignacio Portela, María Magdalena Fló Díaz, Martín Marín Gutiérrez, Mónica Álvarez, Beatriz Turell Novo, Lucía |
| author2_role | author author author author author author author |
| author_browse | Fló Díaz, Martín Lombide, Rodrigo López, Ignacio Marín Gutiérrez, Mónica Portela, María Magdalena Steglich Guarino, Martina Turell Novo, Lucía Álvarez, Beatriz |
| author_facet | Steglich Guarino, Martina Lombide, Rodrigo López, Ignacio Portela, María Magdalena Fló Díaz, Martín Marín Gutiérrez, Mónica Álvarez, Beatriz Turell Novo, Lucía |
| author_role | author |
| collection | COLIBRI |
| dc.contributor.none.fl_str_mv | Steglich Guarino Martina, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. Lombide Rodrigo, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. López Ignacio, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica. Portela María Magdalena, IIBCE Fló Díaz Martín, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica Marín Gutiérrez Mónica, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. Álvarez Beatriz, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. Turell Novo Lucía, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. |
| dc.creator.none.fl_str_mv | Steglich Guarino, Martina Lombide, Rodrigo López, Ignacio Portela, María Magdalena Fló Díaz, Martín Marín Gutiérrez, Mónica Álvarez, Beatriz Turell Novo, Lucía |
| dc.date.none.fl_str_mv | 2020 2022-06-01T13:39:36Z 2022-06-01T13:39:36Z |
| dc.format.none.fl_str_mv | 15 h. application/pdf |
| dc.identifier.none.fl_str_mv | Steglich Guarino, M, Lombide, R, López, I, [y otros] "Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34". PLoS ONE. [en línea] 2020, 15(10): e0240580. 15 h. DOI: 10.1371/journal.pone.0240580 1932-6203 https://hdl.handle.net/20.500.12008/31779 10.1371/journal.pone.0240580 |
| dc.language.none.fl_str_mv | en eng |
| dc.publisher.none.fl_str_mv | Public Library of Science |
| dc.relation.none.fl_str_mv | PLoS ONE, 2020, 15(10): e0240580 |
| dc.rights.none.fl_str_mv | info:eu-repo/semantics/openAccess Licencia Creative Commons Atribución (CC - By 4.0) |
| dc.source.none.fl_str_mv | reponame:COLIBRI instname:Universidad de la República instacron:Universidad de la República |
| dc.subject.none.fl_str_mv | Human serum albumin Antioxidants Protein multimerization Gene expression Chromatography Saccharomycetales |
| dc.title.none.fl_str_mv | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 |
| dc.type.none.fl_str_mv | Artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
| description | Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a noncovalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | anni_093a2ff7b4f149f92f8fde9e4a188fa7 |
| identifier_str_mv | Steglich Guarino, M, Lombide, R, López, I, [y otros] "Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34". PLoS ONE. [en línea] 2020, 15(10): e0240580. 15 h. DOI: 10.1371/journal.pone.0240580 1932-6203 10.1371/journal.pone.0240580 |
| instacron_str | Universidad de la República |
| institution | Universidad de la República |
| instname_str | Universidad de la República |
| language | eng |
| language_invalid_str_mv | en |
| network_acronym_str | anni |
| network_name_str | oai-lr-anni |
| oai_identifier_str | oai:colibri.udelar.edu.uy:20.500.12008/31779 |
| publishDate | 2020 |
| publishDateSort | 2020 |
| publisher.none.fl_str_mv | Public Library of Science |
| reponame_str | COLIBRI |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | Licencia Creative Commons Atribución (CC - By 4.0) |
| spelling | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34Steglich Guarino, MartinaLombide, RodrigoLópez, IgnacioPortela, María MagdalenaFló Díaz, MartínMarín Gutiérrez, MónicaÁlvarez, BeatrizTurell Novo, LucíaHuman serum albuminAntioxidantsProtein multimerizationGene expressionChromatographySaccharomycetalesHuman serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a noncovalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives.Public Library of ScienceSteglich Guarino Martina, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.Lombide Rodrigo, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.López Ignacio, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica.Portela María Magdalena, IIBCEFló Díaz Martín, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química BiológicaMarín Gutiérrez Mónica, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.Álvarez Beatriz, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.Turell Novo Lucía, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.2022-06-01T13:39:36Z2022-06-01T13:39:36Z2020Artículoinfo:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion15 h.application/pdfSteglich Guarino, M, Lombide, R, López, I, [y otros] "Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34". PLoS ONE. [en línea] 2020, 15(10): e0240580. 15 h. DOI: 10.1371/journal.pone.02405801932-6203https://hdl.handle.net/20.500.12008/3177910.1371/journal.pone.0240580reponame:COLIBRIinstname:Universidad de la Repúblicainstacron:Universidad de la RepúblicaenengPLoS ONE, 2020, 15(10): e0240580Las obras depositadas en el Repositorio se rigen por la Ordenanza de los Derechos de la Propiedad Intelectual de la Universidad de la República.(Res. Nº 91 de C.D.C. de 8/III/1994 – D.O. 7/IV/1994) y por la Ordenanza del Repositorio Abierto de la Universidad de la República (Res. Nº 16 de C.D.C. de 07/10/2014)info:eu-repo/semantics/openAccessLicencia Creative Commons Atribución (CC - By 4.0)oai:colibri.udelar.edu.uy:20.500.12008/317792026-04-14T10:09:48Z |
| spellingShingle | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 Steglich Guarino, Martina Human serum albumin Antioxidants Protein multimerization Gene expression Chromatography Saccharomycetales |
| status_str | publishedVersion |
| title | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 |
| title_full | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 |
| title_fullStr | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 |
| title_full_unstemmed | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 |
| title_short | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 |
| title_sort | Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34 |
| topic | Human serum albumin Antioxidants Protein multimerization Gene expression Chromatography Saccharomycetales |
| url | https://hdl.handle.net/20.500.12008/31779 |