Preparation And Application Of Biological Macromolecules Imprinted Polymers For Lysozyme And BSA

Molecular imprinting technology (MIT) is a new technology for high efficiency separation. The MIP synthesized by using target molecule as template shows specific recognition to the imprinted molecule. MIP imprinted polymers have the capability of high specific recognition similar to that of antibody or receptor, which shows specific adsorbability and high selectivity to target protein, and have great application values to the preparation of complex samples, such as proteomics or recombinant protein drugs, etc. In this paper entrapment method was employed to synthesize the molecularly imprinted polymers of lysozyme and bovine serum albumin (BSA) by using lysozyme and BSA as the template molecule. The synthesized polymers were characterized, and their specific adsorption to template molecules lysozyme and BSA was detected respectively and compared with each other. The Lys-MIP and BSA-MIP were also applied to separate and enriched lysozyme in egg white and albumin in serum in Lys and BSA, repectively. In addition, the refolding efficiency of reduction denatured lysozyme was improved by using Lys-MIP assisted protein folding. Meanwihle, Lys-MIP magnetic composite microspheres were also prepared and characterized.This thesis mainly contains five parts as below:1. Introduction:This part mainly introduces the concept, the advances of preparation methods and its application of molecular imprinting technology. The preparation of magnetic macromolecular microspheres and their application in biological analysis also were summuried. In addition, the refolding methods of denatured proteins were also reviewed. In this part, it was cited 104 references.2. Preparation and characterization of molecularly imprinted polymer for lysozyme and BSAWith using lysozyme and BSA as the template molecules and methacrylic acid as functional monomer, lysozyme and BSA molecularly imprinted polymers (Lys-MIP, BSA-MIP) with specific selectivity and recognition for lysozym and BSA, were synthesized using entrapment method, repectively. The products were characterized by scanning electron microscopy(SEM) and infrared spectroscopy(FT-IR). Both of the good selectivity and specific recognition of Lys-MIP and BSA-MIP to the template moclecules were proved with the specific adsorption to the template molecules. With the comparison of the results, it indicated that Lys-MIP showed better imprinting effect than that of BSA-MIP, but imprinted protein with larger molecular weight still has many problems to be discussed.3. The application of molecular imprinted polymers of Lys-MIP and BSA-MIPLys-MIP was used as separation media and filled into a simple column (70mm×10mm ID). Lysozyme from hen egg white was separated and purified, and the purity was 96.3%, and the mass recovery was 98.2%. When BSA-MIP was used to separate bovine serum albumin from bovine serum, the result shows a good adsorption to BSA, but also have adsorption to other proteins, thus, more researches should be carried out to achieve better imprinting effect. When BSA-MIP was employed removing high abundant proteins from cord blood, it shows a good removing effect. The application has great significance for studying low abundance protein in complex samples.4. The preparation and application of lysozyme molecularly imprinted magnetic composite microspheresThe Fe3O4 magnetic microspheres and lysozyme imprinted polymer were combined together, and monomer polymerization method was employed to prepare lysozyme molecularly imprinted magnetic composite microspheres (Lys-MCMs-MIP). The prepared Lys-MCMs-MIP was used for separating lysozyme from hen egg white. Under the action of external magnetic field, lysozyme can be separated very fast and conveniently from hen egg white. Imprinted magnetic composite microspheres can also be used in the enrichment and separation of low abundance proteins, as well as in the removing of media and high abundance proteins in complex samples.5. lysozyme imprinted polymer assisted protein refolding:In this part, the refolding of reduced-denatured lysozyme assisted by lysozyme molecular imprinted polymer was investigated. When Lys-MIP was added into the refolding buffer solution, the refolding of reduced-denatured lysozyme can be enhanced with the comparison of dilution method. The activity recovery can reach to be 90.33%, and the mass recovery to 89.51%, which are higher than that of dilution method. The result shows that the Lys-MIP can be used as refolding helper to improve the renaturation efficiency of denatured protein, and it is very important to the produce of recombinant drugs. In additon, the refolding mechanism of denatured proteins assisted by molecular imprinted polymer was also discussed.