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|標題:||Hydroxyapatite-based immobilized metal affinity adsorbents for protein purification||作者:||Suen, R.B.
|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.||摘要:||
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.
|Appears in Collections:||化學工程學系所|
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