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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.

Abstract：

PURPOSE:Recombinant human serum albumin (rHSA), secreted by a Pichia pastoris expression 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.

journal_name

Pharm Res

journal_title

Pharmaceutical research

authors

Watanabe H,Yamasaki K,Kragh-Hansen U,Tanase S,Harada K,Suenaga A,Otagiri M

doi

10.1023/a:1013391001141

subject

Has Abstract

pub_date

2001-12-01 00:00:00

pages

1775-81

issue

eissn

0724-8741

issn

1573-904X

journal_volume

pub_type

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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.