Preparation Of An Affinity Thermo-sensitive Polymer And Its Application In Affinity Precipitation

Poly N-vinylcaprolactam (PNVCL), one of the temperature-sensitive water-soluble polymers, has caprolactam-ring groups in its side-chain and owns a LCST, which so lies in the range of human body temperature. PNVCL is not only ionic water-soluble, heat sensitivity, but also biocompatible. As containing both coordination atoms of N and O in the polymer chain, PNVCL can be widely used in metal ion affinity chromatography with a variety of metal ions chelated. In addition, it has been also applied in the separation, concentration, immobilization of biological macromolecules and drug release, drug encapsulation.Affinity precipitation is a separation and purification technology combined biological affinity interaction with precipitation of biological macromolecules. It is considered effectiveness for the isolation and purification of enzymes and other proteins. Large-scale, recycling of affinity ligand and specific isolation of pure enzymes and other target protein molecules can also be achieved.This investigation synthesized a thermo-sensitive copolymer PNVCL-co-MAA by free radical polymerization of N-vinylcaprolactam (NVCL) and methyl methacrylate (MAA). Carboxyl group was introduced by copolymeriazation NVCL with MAA and then copper ion was chelated to the thermo-sensitive copolymer. Therefore, the cation-loaded copolymer obtained both temperature-sensitivity and affinity, and could be applied to the affinity precipitation of proteins.The main contents include:Using water as a reaction solvent, oxidation-reduction system as initiator system, NVCL and MAA as monomer, synthesized the temperature-sensitive polymer PNVCL-co-MAA. The optimal conditions for synthesis are:NVCL concentration of 4%(w/v), MAA concentration of 0.4%(v/v), initiator APS concentration of 0.16% (w/v). The thermo-sensitive polymer prepared has good temperature-sensitive, and was suitable for protein separation and purification. PNVCL-co-MAA-Cu, the macromolecular thermo-sensitive affinity ligand, was synthesized by binding the copper ion as the metal affinity ligand. Results showed that the optimal chelating condition was:pH at 5.0,2.0 mg/ml of initial concentration of copper ions,1 hour of chelating time. The maximum amount of loaded copper ion was obtained of 48.1 mg per gram of temperature-sensitive polymer PNVCL-co-MAA, detected by the Ultraviolet Spectrophotometry. The affinity temperature-sensitive polymer PNVCL-co-MAA-Cu prepared has good storage-stability in both acidic and neutral environment.Factors which may affect the precipitation and elution of BSA, which was studied as the model protein, using PNVCL-co-MAA-Cu were studied. The optimum precipitation conditions are:pH at 6.0, temperature at 10℃, initial concentration of BSA of 1.0 mg/ml. The sequence of three different thermo-sensitive polymers with respect to increasing of the amount of BSA precipitated was PNVCL< PNVCL-co-MAA<PNVCL-co-MAA-Cu. The results showed that, the interaction between BSA molecules and affinity temperature-sensitive polymer PNVCL-co-MAA-Cu was specific. The optimum elution conditions are:pH at 4.0, 0.20 M NaCl and 0.06 M EDTA contained in 0.05 M Tris-HCl buffer.The affinity temperature-sensitive polymer PNVCL-co-MAA-Cu can be recycled many times, saving a lot of resources. It is an efficient, environmental friendly affinity separation media.