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標題: Hydroxyapatite-based immobilized metal affinity adsorbents for protein purification
作者: Suen, R.B.
Lin, S.C.
Hsu, W.H.
關鍵字: immobilized metal affinity chromatography;hydroxyapatite;protein;purification;aqueous 2-phase systems;ion affinity;chromatography;acid;2-epimerase;adsorption;extraction;mechanism;sediments;matrices
Project: Journal of Chromatography A
期刊/報告no:: Journal of Chromatography A, Volume 1048, Issue 1, Page(s) 31-39.
The employment of metal ion-charged hydroxyapatite for the one-step purification of poly(His)-tagged recombinant proteins was investigated. Fe(III) showed the highest selectivity toward the poly(His)-tagged D-hydantoinase and the best operation stability. The optimal selectivity was observed in 20 mM pH 8.0 buffer containing 150 mM NaCl and 50 mM NaF. The adsorbed poly(His)-tagged enzyme could be quantitatively recovered from hydroxyapatite with 150 mM pH 8.0 phosphate buffer. The capacity of Fe(Ill)-loaded hydroxyapatite for poly(His)-tagged D-hydantoinase was 4.9 mg/g hydroxyapatite, comparable to commercial agarose-based Ni-NTA adsorbents. Under optimal conditions, a D-hydantoinase preparation with a purity above 95% from crude cellular lysate could be obtained with the one-step purification process employing Fe(Ill)-loaded hydroxyapatite. The application of Fe(Ill)-loaded hydroxyapatite for the purification of poly (His)-tagged N-acetyl-D-glucosamine 2-epimerase under denaturing conditions was also demonstrated. These results demonstrate that hydroxyapatite is a promising adsorbent for immobilized metal affinity chromatography. (C) 2004 Published by Elsevier B.V.
ISSN: 0021-9673
DOI: 10.1016/j.chroma.2004.06.132
Appears in Collections:化學工程學系所

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