Título:
Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34
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Autor:
Steglich Guarino, Martina
Lombide, Rodrigo
López, Ignacio
Portela, María Magdalena
Fló Díaz, Martín
Marín Gutiérrez, Mónica
Álvarez, Beatriz
Turell Novo, Lucía
Lombide, Rodrigo
López, Ignacio
Portela, María Magdalena
Fló Díaz, Martín
Marín Gutiérrez, Mónica
Álvarez, Beatriz
Turell Novo, Lucía
Tutor:
Tipo de documento:
Artículo
Editor:
Permyakov, E. A.
Palabras clave:
Human serum albumin
Antioxidants
Protein multimerization
Gene expression
Chromatography
Saccharomycetales
Antioxidants
Protein multimerization
Gene expression
Chromatography
Saccharomycetales
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Año de publicación:
2020
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Resumen:
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.
Descripción:
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Public Library of Science
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PLoS ONE, 2020, 15(10): e0240580
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Citación:
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
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ISSN:
1932-6203
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Licencia:
Licencia Creative Commons Atribución (CC - By 4.0)
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| Ficheros | Descripción | Tamaño | Formato | ||
|---|---|---|---|---|---|
| 10.1371journal.pone.0240580.pdf | — | 1.5 MB | Adobe PDF |