Construction of expression system for human α1-acid glycoprotein in Pichia pastoris and evaluation of its drug-binding properties
Construction of expression system for human α1-acid glycoprotein in Pichia pastoris and evaluation of its drug-binding properties
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DOI：
10.1124/dmd.104.000513
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发表时间：
2004-10-01
影响因子：
3.9
通讯作者：
Otagiri, M
中科院分区：
文献类型：
--
作者：
Nishi, K;Fukunaga, N;Otagiri, M
人α 1-酸性糖蛋白（hAGP）是一种血浆糖蛋白，作为碱性配体的主要载体。这是重组hAGP（rhAGP）的首次报道。在本研究中，rhAGP在甲醇酵母表达载体，pPIC 9的巴斯德毕赤酵母（GS115），表达，然后通过阴离子交换，疏水相互作用，和凝胶过滤层析纯化。rhAGP的分子量远低于hAGP，这是由于糖链含量的差异。糖肽酶F酶切结果表明rhAGP与hAGP的肽段相同。圆二色谱分析结果表明，rhAGP主要形成富含β折叠的结构，与hAGP相同，是典型的脂质运载蛋白家族。从使用AGP结合药物（氯丙嗪、华法林和孕酮）和喹哪啶红作为结合位点探针的实验中，表明rhAGP也具有与hAGP相同的配体结合能力和结合位点结构。这些结果有力地表明，这种重组hAGP（rhAGP）是非常有用的探索hAGP的配体结合位点和生物学功能。
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.