The Development And Application Of New Chromatographic Refolding Of Proteins By Dye Affinity Chromatography

There are three parts in this thesis. A novel column-based protein refolding strategy was developed using dye-ligand affinity chromatography based on macroporous biomaterial in the first chapter. A simple strategy was proposed for preparation of spherical silica-supported porous chitosan matrix based on sol–gel reaction and simple treatment with ammonia solution in the second chapter. Spherical silica-supported biosorbent for copper ions removal in wastewater was developted by sol–gel reaction and simple treatment with sodium hydroxide in the third part.1. A novel column-based chromatographic protein refolding strategy was developed using dye–ligand affinity chromatography (DLAC) based on macroporous biomaterial. Chitosan–silica (CS–silica) biomaterial with macroporous surface was used as the supporting matrix for the preparation of the DLAC material. The dye ligand Cibacron Blue F3GA (CBF) was selected as affinity handle and could be covalently immobilized to form dye-ligand affinity adsorbent (CBF–CS–silica) using the reactivity of -NH2 on CS–silica biomaterial. After the model protein catalase was denatured with 6 mol/L urea, the denaturant could be rapidly removed and catalase could be successfully refolded as facilitated by the adsorption of CBF–CS–silica. The urea denaturation process and elute condition for the chromatographic refolding were optimized by measuring tryptophan fluorescence and activity of catalase. The refolding performance of the proposed DLAC was comparable to dilution refolding. The protein concentration during the proposed chromatographic refolding was increased by a factor of 20 without reducing the yield achieved compared to dilution refolding. The column-based protein refolding strategy based on dye affinity chromatography with porous biomaterial being matrix possessed potential in chromatographic refolding of protein. 2. A new and effective strategy was proposed for the preparation of organic-inorganic composite matrix by using spherical silica as supporting core and porous chitosan (CS) hybrid layer as shell based on sol–gel reaction and simple treatment with ammonia solution. After metal ion loading, immobilized metal affinity adsorbent for protein adsorption was obtained. Scanning electron microscopy investigation showed that the wet phase-inversion of CS in ammonium hydroxide solution endowed the coated CS hybrid layer with chemically and mechanically stabilized pore structure. X-Ray diffraction investigation revealed significant decrease of CS crystallization, indicating the availability of active amine groups. The as-prepared matrix was also characterized using simultaneous thermogravimetry and differential scanning calorimetry. After loading Cu2+ as pseudo-biospecific ligand, new immobilized metal affinity adsorbent was obtained and its protein adsorption performance was evaluated by batch adsorption experiments using bovine serum albumin (BSA) as a simple model protein. The affinity adsorbent showed fast kinetics and high capability for BSA adsorption. The proposed approach and the prepared matrix showed potential as a platform to conduct bioanalysis.3. A new and effective strategy was proposed for preparing new organic-inorganic composite biosorbent by using spherical silica as supporting core and chitosan (CS)-based hybrid layer as shell based on sol–gel reaction and simple treatment with sodium hydroxide (NaOH). The coating layer was covalently bound on the supporting silica through polysaccharide incorporated sol–gel process starting from CS and inorganic precursorγ-glycidoxypropyltrimethoxysiloxane (GPTMS). GPTMS had epoxide groups and cross-linked amine groups of CS to avoid its acidic solubility. The composite biosorbent had coarse surface due to the wet phase-inversion by treating in NaOH solution. The prepared biosorbent could be used in treating electric plating wastewater.