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In Vitro and in Vivo Properties of Recombinant Human Serum Albumin from Pichia Pastoris Purified by a Method of Short Processing Time

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Abstract

Purpose. Recombinant human serum albumin (rHSA), secreted by a Pichia pastorisexpression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed.

Methods. Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with 125I-labeled albumin.

Results. Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations.

Conclusion. The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

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Authors and Affiliations

  1. Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan

    Hiroshi Watanabe, Keishi Yamasaki, Ayaka Suenaga & Masaki Otagiri

  2. Department of Medical Biochemistry, University of Aarhus, DK-8000, Aarhus C, Denmark

    Ulrich Kragh-Hansen

  3. Department of Biochemistry, School of Medicine, Kumamoto University, Honjo 2-2-1, Kumamoto, 860-0811, Japan

    Sumio Tanase

  4. Department of Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto, 862-0973, Japan

    Kumiko Harada

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  1. Hiroshi Watanabe
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Watanabe, H., Yamasaki, K., Kragh-Hansen, U. et al. In Vitro and in Vivo Properties of Recombinant Human Serum Albumin from Pichia Pastoris Purified by a Method of Short Processing Time. Pharm Res 18, 1775–1781 (2001). https://doi.org/10.1023/A:1013391001141

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