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Drug Metabolism and Disposition Fast Forward
First published on June 30, 2004; DOI: 10.1124/dmd.104.000513

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Received for publication May 4, 2004.
Revised June 24, 2004.
Accepted for publication June 25, 2004.

CONSTRUCTION OF EXPRESSION SYSTEM FOR HUMAN {alpha}1-ACID GLYCOPROTEIN IN PICHIA PASTORIS AND EVALUATION OF ITS DRUG-BINDING PROPERTIES

Koji Nishi 1, Naoko Fukunaga 1, Masaki Otagiri 1*

1 Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, kumamoto university

* Address correspondence to: E-mail: otagirim{at}gpo.kumamoto-u.ac.jp

Abstract

Human {alpha}1-acid glycoprotein (hAGP) is a plasma glycoprotein that functions as a major carrier of basic ligands. This is the first report for the recombinant hAGP (rhAGP). In this study, rhAGP was expressed in the methylotropic yeast Pichia pastor(GS115) using the expression vector, pPIC9, and then purified by anionic exchange, hydrophobic interaction and gel filtration chromatography. The molecular weight of rhAGP was much lower than that of hAGP, because of the difference in glycan chain content. Results of glycopeptidase F (GPF) digestion suggest that the peptide moiety of rhAGP was the same as that of hAGP. The results of CD spectra measurement indicated that rhAGP predominantly formed a {beta}-sheet rich structure that was the same as that of hAGP and typical of the lipocalin family. From the experiments using AGP-binding drugs (chlorpromazine, warfarin and progesterone) and quinaldine red as a probe for binding site, it was indicated that rhAGP also had the same ligand-binding capacity and the binding site structure as hAGP. These findings strongly suggest that this rhAGP is very useful for the exploration of the ligand-binding site and biological function of hAGP.


Key words: drug disposition, drug interactions, protein binding, recombinant proteins


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M. Katsuki, V. T. G. Chuang, K. Nishi, K. Kawahara, H. Nakayama, N. Yamaotsu, S. Hirono, and M. Otagiri
Use of Photoaffinity Labeling and Site-directed Mutagenesis for Identification of the Key Residue Responsible for Extraordinarily High Affinity Binding of UCN-01 in Human {alpha}1-Acid Glycoprotein
J. Biol. Chem., January 14, 2005; 280(2): 1384 - 1391.
[Abstract] [Full Text] [PDF]


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