Adsorption behaviors of recombinant proteins on hydroxyapatite-based immobilized metal affinity chromatographic adsorbents

The equilibrium adsorption of three homo-oligomeric model recombinant proteins containing up to 8 poly(histidine) affinity tags on a hydroxyapatite-based immobilized metal affinity chromatography (IMAC) adsorbent is reported in this study. The experimental data are well fitted with the three-parameter Langmuir–Freundlich isotherm model, indicating the presence of positive cooperativity for the adsorption of these model proteins. The maximum capacity and the binding affinity of the IMAC adsorbent for the model proteins are in principle dependent on the size and the number of affinity tags of proteins, respectively. The exceptionally high association constant of the octameric racemase, probably due to simultaneous multipoint attachment, make it difficult to elute racemase from the adsorbent. The adsorption isotherms under denaturing conditions are well fitted with the Langmuir model. Results of Scatchard analysis further suggest the homogeneous adsorption of the model protein subunits under denaturing conditions. The binding capacities and affinities of the adsorbent under denaturing conditions for the three unfolded protein subunits become essentially identical because the molecular size and number of poly(His) tags of the unfolded polypeptide chains of the three protein subunits are the same. The significant reduction in association constants under denaturing conditions suggests that high concentration of urea could interfere with the binding of proteins on the hydroxyapatite-based adsorbent.