Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34

Abstract

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.

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“…HSA includes three major domains, according to high-resolution X-ray crystallographic structures, and is the most abundant protein in plasma, accounting for approximately 60% of total plasma proteins, and it is known for being responsible for several functions. Some of these include maintaining colloid osmotic pressure and binding and transporting a wide range of ligands, including fatty acids, bilirubin, hormones, metal ions, and medicines [55,56]. An important application for this protein is also found in cell differentiation, especially in recent years, and significant studies have been carried out on processes such as the regulation of osteogenic differentiation [57].…”

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