Artículo
Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34
Fecha
2020Registro en:
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
Autor
Steglich Guarino, Martina
Lombide, Rodrigo
López, Ignacio
Portela, María Magdalena
Fló Díaz, Martín
Marín Gutiérrez, Mónica
Álvarez, Beatriz
Turell Novo, Lucía
Institución
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.