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|標題:||Equilibrium adsorption of poly(His)-tagged proteins on immobilized metal affinity chromatographic adsorbents|
Immobilized metal affinity chromatography
|摘要:||A systematic analysis on the adsorption of homo-oligomeric, poly(His)-tagged recombinant proteins on a hydroxyapatite-based immobilized metal affinity chromatography adsorbent was performed. Under non-denaturing conditions, the Langmuir-Freundlich isotherm model was found ideal for predicting the adsorption behavior of the model proteins. The dissociation constants, as low as 10−9 M, decreased with the number of poly(His) tags, suggesting the presence of multi-point attachment. The maximum adsorption capacities, ranging from 79.1 nmol/g for the 88 kDa epimerase to 42.8 nmol/g for the 320 kDa racemase, were inversely proportional to the contact surface areas of the proteins. Under denaturing conditions, the Langmuir isotherm model fitted well with the experimental data. The maximum adsorption capacities for and the dissociation constants of the three model proteins were essentially identical, as the subunits of the model proteins were of similar dimensions and behaved similarly in the absence of complex tertiary or quaternary structure. Phosphate buffer at a concentration of 500 mM, pH 8.0 was found to be effective for the elution of the model proteins with a recovery yield more than twofold higher than that obtained with 500 mM imidazole. The results suggest the hydroxyapatite-based adsorbent is a promising alternative for large scale applications.|
|Appears in Collections:||化學工程學系所|
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