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標題: Applications of hydroxyapatite-based immobilization metal affinity adsorbents for the purification of recombinant proteins
作者: 孫潤伯
關鍵字: 氫氧基磷灰石;Hydroxyapatite;固定金屬親和層析法;蛋白質純化;Immobilized metal ion affinity chromatography;protein purification
出版社: 化學工程學系
In this study, the application of hydroxyapatite as IMAC absorbent for the purification of soluble recombinant poly(His)-tagged D-hydantoinase and 2-epimerase was investigated. Fe3+ as the coordinating metal ion showed the best adsorption selectivity. The addition of 0.15~0.25M NaCl and 0.05M NaF can increase D-hydantoinase recovery and activity. Changes in pH and concentration of sodium phosphate buffer can wash nonspecifically adsorbed proteins and elute D-hydantionase from adsorbent effectively. The recovery yield and purity of D-hydantoinase were 40% and above 95% respectively, under optimal purification conditions. The capacity of hydroxyapatite-based IMAC absorbent was 9.92 mg/g HAP for total proteins, and 4.9 mg/g HAP for D-hydantoinase, The absorption of denatured 2-epimerase demonstrate that hydroxyapatite-based IMAC absorbent can be applied to purification of recombinant poly(His)-tagged protein under denaturing conditions.

本研究探討以氫氧基磷灰石做為固定化金屬親和吸附材及其於基因重組 D-hydantoinase(DHTase)純化之應用。最適回收純化程序: 配位金屬離子為Fe3+,吸附緩衝液 20mM Tris-HCl 0.25M NaCl 0.05M NaF pH 7.5,清洗緩衝液0.1M sodium phosphate pH 7.0,脫附緩衝液0.15M sodium phosphate pH 8.0。在最適條件時,總蛋白質吸附量為9.92 mg/g HAP,D-hydantoinase 吸附量為4.9 mg /g HAP,活性回收率為40%,純度為≧95%。研究結果顯示,以Fe3+為配位金屬離子有最佳的吸附選擇性,pH 7.5可以提升酵素回收率與活性,氯化鈉與氟化鈉的添加可以減少非特定吸附產生與增加回收率。由2-epimerase的吸附則證明了此吸附材於基因重組蛋白質的吸附再現性,以及吸附純化程序於失活條件下亦可進行。
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