Preparation of an Affinity Thermo-sensitive Polymer and Its Application in Affinity Precipitation

Poly-N-vinyl caprolactam (PNVCL) is a side-chain is ring caprolactam group of water-soluble temperature sensitive polymer and its LCST just in human physiological temperature range. It not only has the ionic water-soluble, heat-sensitive, but also biocompatible. Ligand containing both N and O atoms in the polymer on the macromolecular chains, it can be with a variety of metal ions can form a stable chelate can be widely applied to the metal ion affinity chromatography. In addition, it is in the separation of biological macromolecules, concentrated, immobilized and drug-embedded, and drug release. Affinity precipitation is a combination of biological affinity interactions with the precipitation separation of biological macromolecules of the protein separation and purification techniques, the method is considered to be an effective means of Separation and Purification of Enzymes and other proteins. Its advantage is recovered using the protein affinity ligands can be many times more economical, can achieve large scale, rapid and specific separation of the purified enzyme and other target protein molecule. This thesis uses the method of copolymerization - chelated metal ion, N-vinyl caprolactam (NVCL) monomers and methacrylic acid (MAA) to radical copolymerization, the polymer both having a temperature sensitive properties, and also provides carboxyl and metal ion chelation reaction. Chelate divalent copper ion, then PNVCL-co-MAA copolymer thereby obtained also has temperature sensitivity and affinity for the metal chelate affinity ligands, and applied to a protein affinity precipitation. The main contents include: with water as the reaction solvent, oxidation - reduction system as a reaction initiator the system, NVCL and MAA monomer synthesized temperature sensitive polymer PNVCL-co-of MAA, the optimal conditions for its synthesis: NVCL concentration of 4% (% of the total mass of the solution), MAA concentration of 0.4% (of the total volume of the solution), the initiator APS dosage 0.16% (% of the total mass of the solution), prepared by the temperature-sensitive polymer has better thermal stimuli carrier, suitable as a protein purification. Then copper ions, was prepared by the protein macromolecules affinity with temperature-sensitive the base PNVCL-co-MAA-Cu through chelate fund case. Experimental optimization, the results show that the best ligand chelate conditions: pH = 5.0, and the optimum copper ion initial concentration of 2.0 mg / ml, for 1 hour. UV spectroscopy detection temperature sensitive polymer PNVCL-co-MAA copper ion maximum adsorption capacity of 48.1mg / g. This thesis is prepared pro-and thermo-sensitive polymer PNVCL-co-MAA-Cu has good storage stability in acidic and neutral environment, a wide range of solvents applicable. BSA as a model protein of pro and temperature sensitive polymer PNVCL-co-MAA-Cu on the amount of precipitation and elution volume of BSA conditions, and optimize them to get the best precipitation conditions: pH6 .0, and the temperature was 10 ° C, the initial concentration of BSA was 1.0 mg / ml. Three different temperature-sensitive polymer, the comparison of the amount of precipitation of BSA at the same concentration of BSA: PNVCL lt; PNVCL-co-MAA lt; PNVCL-co-MAA-Cu. Experimental results show that the specificity of the interaction between the BSA molecules and pro-and temperature-sensitive polymer PNVCL-co-MAA-Cu. The preferred elution conditions for affinity and temperature sensitive polymer PNVCL-co-MAA-Cu for BSA: pH 4.0,0.05 M Tris-HCl buffer, containing 0.20 M NaCl and 0.06 M EDTA. Renewable experiments show of pro and temperature sensitive polymer PNVCL-co-MAA-Cu can be repeatedly recycled, saving a lot of resources, is an environmentally friendly, efficient performance affinity separation media.