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Equilibrium adsorption of poly(His)-tagged proteins on immobilized metal affinity chromatographic adsorbents

Paper ID Volume ID Publish Year Pages File Format Full-Text
3825 191 2011 9 PDF Available
Title
Equilibrium adsorption of poly(His)-tagged proteins on immobilized metal affinity chromatographic adsorbents
Abstract

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.

Research highlights► Selective adsorption of recombinant proteins with the adsorbent is demonstrated. ► Adsorption of proteins is best described by the Langmuir–Freundlich model. ► Adsorption of denatured proteins can be described by the Langmuir model. ► Adsorption is mediated predominantly by affinity-like interations. ► Affinity of proteins is proportional to the number of accessible poly(His) tags.

Keywords
Hydroxyapatite; Adsorption isotherm; Immobilized metal affinity chromatography; Langmuir; Langmuir–Freundlich
First Page Preview
Equilibrium adsorption of poly(His)-tagged proteins on immobilized metal affinity chromatographic adsorbents
Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 54, Issue 1, 15 March 2011, Pages 1–9
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering