Construction of expression system for human alpha 1-acid glycoprotein in Pichia pastoris and evaluation of its drug-binding properties

Drug Metabolism and Disposition : the Biological Fate of Chemicals
Koji NishiMasaki Otagiri

Abstract

Human alpha1-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 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 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.

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Citations

May 6, 2008·Biochimica Et Biophysica Acta·Ferenc ZsilaGyörgy Mády
Sep 30, 2006·Biochimica Et Biophysica Acta·Ilona FitosMiklós Simonyi
Nov 18, 2005·Bioorganic & Medicinal Chemistry·Eszter HazaiMiklós Simonyi
Nov 8, 2005·Drug Metabolism and Pharmacokinetics·Masaki Otagiri
Aug 28, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Aleksandra OwczarzyMałgorzata Maciążek-Jurczyk

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