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Research ArticleArticle

CONSTRUCTION OF EXPRESSION SYSTEM FOR HUMAN α1-ACID GLYCOPROTEIN IN PICHIA PASTORIS AND EVALUATION OF ITS DRUG-BINDING PROPERTIES

Koji Nishi, Naoko Fukunaga and Masaki Otagiri
Drug Metabolism and Disposition October 2004, 32 (10) 1069-1074; DOI: https://doi.org/10.1124/dmd.104.000513
Koji Nishi
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Naoko Fukunaga
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Masaki Otagiri
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Abstract

Human α1-acid glycoprotein (hAGP) is a plasma glycoprotein that functions as a major carrier of basic ligands. This is the first report of the recombinant hAGP (rhAGP). In this study, rhAGP was expressed in the methylotropic yeast Pichia pastoris (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 digestion suggest that the peptide moiety of rhAGP was the same as that of hAGP. The results of circular dichroism spectra measurement indicated that rhAGP predominantly formed a β-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 the binding site, it was indicated that rhAGP also had the same ligand-binding capacity and binding site structure as hAGP. These findings strongly suggest that this recombinant hAGP (rhAGP) is very useful for the exploration of the ligand-binding site and biological function of hAGP.

Footnotes

  • doi:10.1124/dmd.104.000513.

  • ABBREVIATIONS: AGP, α1-acid glycoprotein; hAGP, human AGP; rhAGP, recombinant hAGP; CD, circular dichroism; GPF, glycopeptidase F; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; MD, minimal dextrose; MM, minimal methanol; Mut, methanol utilization; Mut+, high methanol utilization; MutS, slow methanol utilization; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

    • Received May 4, 2004.
    • Accepted June 25, 2004.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 32 (10)
Drug Metabolism and Disposition
Vol. 32, Issue 10
1 Oct 2004
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Research ArticleArticle

CONSTRUCTION OF EXPRESSION SYSTEM FOR HUMAN α1-ACID GLYCOPROTEIN IN PICHIA PASTORIS AND EVALUATION OF ITS DRUG-BINDING PROPERTIES

Koji Nishi, Naoko Fukunaga and Masaki Otagiri
Drug Metabolism and Disposition October 1, 2004, 32 (10) 1069-1074; DOI: https://doi.org/10.1124/dmd.104.000513

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Research ArticleArticle

CONSTRUCTION OF EXPRESSION SYSTEM FOR HUMAN α1-ACID GLYCOPROTEIN IN PICHIA PASTORIS AND EVALUATION OF ITS DRUG-BINDING PROPERTIES

Koji Nishi, Naoko Fukunaga and Masaki Otagiri
Drug Metabolism and Disposition October 1, 2004, 32 (10) 1069-1074; DOI: https://doi.org/10.1124/dmd.104.000513
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