İmmobilize Metal Afinite Kromatografisi İçin TiO2 Bazlı Sabit Fazların Sentezi ve Biyomolekül İzolasyonunda Kullanımı

Abstract

A titania based sorbent was developed for His-tagged protein purification by immobilized metal affinity chromatography (IMAC). The synthesis of monodisperse porous titania microbeads ca 5 m in size was performed by a staged shape template sol-gel method using poly(3-chloro-2-hydroxypropyl methacrylate-co-ethylene glycol dimethacrylate) poly(HPMA-Cl-co-EDMA) microbeads as the template material. Iminodiacetic acid-3-glycidoxypropyl trimethoxysilane complex (IDA-GLYMO) was covalently attached onto the titania microbeads via silanization reaction. Ni2+ ions were then attached to the derivatized microbeads via metal-chelate complex formation with the carboxyl groups on the microbeads. The developed sorbent was used for the isolation of histidine-rich proteins in batch affinity system. For this purpose, the isolation of bovin hemoglobin (BHb) as a histidine-rich protein and bovine serum albumin (BSA) as a reference protein were comparatively investigated using Ni2+ attached-monodisperse-porous titania microbeads as the sorbent in batch fashion. The maximum equilibrium BHb adsorption was determined as 140 mg BHb per g sorbent. BHb could be isolated with quantitative desorption yield in the aqueous buffer medium. Although the selected reference, BSA exhibited a limited equilibrium adsorption, it could not be eluted from the sorbent with high yield. The developed sorbent was also used for isolation of hemoglobin from human blood in micro-immobilized metal affinity chromatography (MIMAC) systems in batch and continuous system, and Hb was isolated with ≥95 and 85% purity, respectively, in batch and MIMAC systems. The results show that the developed sorbent allows isolation with high selectivity and high efficiency for histidine-rich protein BHb.