Gelatin-Immobilised Poly(hydroxyethyl methacrylate) Cryogel for Affinity Purification of Fibronectin

Abstract

In this work, fibronectin purification from human plasma with the gelatin-immobilised poly(hydroxyethyl methacrylate) (PHEMA) cryogel has been evaluated. The PHEMA cryogel was prepared by cryo-polymerisation which proceeds in an aqueous solution of monomer frozen inside a plastic syringe. The PHEMA cryogel contained interconnected macrochannels of 10-200μm in diameter. Gelatin molecules were covalently immobilised onto the PHEMA cryogel via carbodiimide activation. The gelatin-immobilised PHEMA cryogel was used to purify fibronectin from human plasma. Fibronectin adsorption from human plasma on the PHEMA cryogel was 0.30mg/ml, while much higher adsorption values, up to 38mg/ml, was obtained with the gelatin-immobilised PHEMA cryogel. The fibronectin adsorption capacity of the gelatin-immobilised PHEMA cryogel did not change with an increase in the flow rate of plasma. Up to 92% of the adsorbed fibronectin was eluted using 2M urea containing 1M NaCl as elution agent. The adsorption-elution cycle was repeated ten times using the same PHEMA cryogel. No remarkable decrease was detected in the adsorption capacity of the gelatin-immobilised PHEMA cryogel.