Preparation Of Basic Protein Molecule Surface-imprinted Materials And Study On Its Macromolecule Recognition Character

Basic protein(isoelectric point, pI>7) is an important part of tissue protein and cell protein, and plays an important biological function in the living body and has important research and applications in the biomedical field. Isolated from biological tissue extraction or using genetic engineering to restructuring synthesis and purification is two main ways to obtain the basic protein. But no matter what method is used, we are in urgent need of development of new separation and purification technology and separation materials. In the purification of materials, molecular imprinted polymers(MIPs) is by far the best choice recognition separating media. At present, the molecularly imprinted polymers(MIPs) as the solid adsorbent, molecularly imprinted solid-phase extraction(MIPSE) technology is widely used in the field of efficient separation and purification of various small molecules and has potential application prospect in biological macromolecular separation and purification.However, the protein molecular imprinting facing many difficulties, basic protein macromolecular surface imprinted material rarely reported. In this study,through molecular design, for the first time basic protein surface imprinted material with high performance was prepared successfully in aqueous medium by using the advanced surface imprinting technique of “synchronously graft-polymerizing and imprinting” and with Trypsin(TRY) and papain(Papain) two kinds of basic protein as the model protein, Sodium p-Styrene Sulfonate(SSS)and Methacrylic acid(MAA) as functional monomer, crosslinked polyvinyl alcohol(CPVA)microspheres as matrix. And On this basis, the recognition features and mechanism of the imprinting materials for template protein molecules is studied in depth. In this paper, the research results in blotting techniques and theoretical aspects of biological macromolecules surface have obvious innovation, and also provides a high-performance surface imprinted material for the efficient separation and purification of basic proteins by using MIPSE technology and has important scientific value in the separation and purification of biologicalmacromolecules and other related biochemical field.First, the interaction between SSS and MAA monomers with basic protein between TRY and Papain were investigated to study by using of UV spectroscopy and fluorescence spectroscopy. And the results show that there is a strong electrostatic interaction between two anionic monomer and basic protein in aqueous medium and so it will automatically form the host-guest complex. Which lay adequate theoretical basis for the implementation of the TRY and Papain surface imprinted.The reaction between methyl acryloyl chloride and the hydroxyl group of CPVA microspheres was made to be carried, resulting in the modified microspheres MAO-CPVA, on which a great deal of polymerizable double bonds, methylacryloyl(MAO) group, were introduced. In aqueous solution, by the initiating of the free radicals produced by persulfate initiating system, the graft-crosslinking polymerization of the monomer SSS around the template TRY and crosslinker N,N’-methylene bisacrylamide(MBA) and double bond on the surface of the microsphere MAO – CPVA was conducted, which produce graft crosslinking polymerization( "Grafting through" grafting), while the template TRY wrapped in the crosslinked network, and so as to achieve the graft polymerization of SSS and TRY surface imprinting and obtaining TRY surface-imprinted material MIP-PSSS/CPVA after removing the template macromolecules. The product microspheres were characterized through infrared spectroscopy(IR) and scanning electron microscopy(SEM). By static combination and competition experiments, the recognition performance and recognition mechanism of surface imprinted microsphere were investigated in depth and the main factors were studied that affect surface imprinting micro identification of selective. The research results show that surface imprinting microspheres MIP- PSSS/CPVA have excellent binding affinity for TRY and posses specific recognition selectivity towards TRY. The binding capacity reaches to 85.9mg/g and relative another basic protein, LZM, the coefficient of selectivity of imprinted microspheres for TRY actually gets up to 17.52. The main factors affecting TRY imprinted microsphere recognition performance are the ratio of the amount of substance of the monomer SSS and the template TRY and of the monomer SSS and the crosslinking agent MBA. Thesuitable mass ratios are 600: 1 and 35: 1. In addition, the TRY imprinting microspheres has a good desorption performance(desorption rate of 98.46%) and re-use performance.Then, with the MAA as functional monomer and the same experimental procedure as described above, the surface imprinted of TRY macromolecules is implemented and obtaining TRY surface-imprinted material MIP-PMAA/CPVA. With Papain as the reference protein, the macromolecular recognition performance of the surface imprinted microsphere is explored.The research results show that surface imprinting microspheres MIP- PMAA/CPVA have excellent binding affinity for TRY and posses specific recognition selectivity towards TRY.The binding capacity reaches to 80.4 mg/g and relative another protein Papain, the coefficient of selectivity of imprinted microspheres for TRY actually gets up to 21.62. The suitable mass ratios of the monomer MAA and the template TRY and of the monomer MAA and the crosslinking agent MBA are 500: 1 and 35: 1.In this study, with Papain as the template protein, another basic protein surface imprinting microsphere was obtained. The nucleophilic substitution reaction between chlorine amine and the hydroxyl group of CPVA microspheres was made to be carried, resulting in the modified microspheres EA-CPVA, on which a great deal of ethyl amino(EA) were introduced.A lot of free radicals are produced on the CPVA microsphere surface through surface oxidation- reduction systems between the amino group on the modified microspheres EA-CPVA and the persulfate in aqueous solution. The graft-crosslinking polymerization( "Grafting through" grafting) of the monomer SSS around the template Papain and crosslinker N,N’-methylene bisacrylamide(MBA) was conducted by these free radicals. While the template Papain wrapped in the crosslinked network, and so as to achieve the graft polymerization of SSS and Papain surface imprinting and obtaining Papain surface-imprinted material MIP-PSSS/CPVA after removing the template. Based on the imprinted microspheres were on the well-characterized, the imprinted microsphere macromolecules recognition performance and recognition mechanisms were studied in depth. Experimental results show that imprinting microspheres MIP- PSSS/CPVA have excellent binding affinity for Papain and posses specific recognition selectivity towards Papain. The binding capacity reaches to44.9 mg/g and relative another protein TRY, the coefficient of selectivity of imprinted microspheres for Papain gets up to 14.35. The suitable mass ratios of the monomer SSS and the template Papain and of the monomer SSS and the crosslinking agent MBA are 750: 1 and45: 1. And the Papain imprinting microspheres MIP- PSSS/CPVA also has a good desorption performance(desorption rate of 98.17%) and re-use performance.Then, with the MAA as functional monomer again and the surface-initiated graft polymerization as described above, the surface imprinted of Papain macromolecules is implemented on the surface of the modified microspheres EA-CPVA and obtaining Papain surface-imprinted material MIP-PMAA/CPVA. The imprinted microspheres MIP-PMAA/CPVA also have excellent binding affinity for Papain and posses specific recognition selectivity towards Papain. The binding capacity reaches to 46.3 mg/g and relative another protein TRY, the coefficient of selectivity of imprinted microspheres for Papain gets up to 17.4. The suitable mass ratios of the monomer MAA and the template Papain and of the monomer MAA and the crosslinking agent MBA are 600: 1 and 40: 1.Finally, the graft anionic polyelectrolyte brush CPVA-g-PSSS was prepared in this research, and the adsorption properties, adsorption mechanism and adsorption thermodynamics of the graft brush to TRY and LZM, two basic protein, were researched. The results show that the anionic polyelectrolyte brush CPVA-g-PSSS possesses very strong adsorption ability for TRY and LZM by the driving of strong electrostatic interaction in aqueous medium. The anionic polyelectrolyte brush is an excellent protein adsorption material, and when protein molecules embedded at full stretch of polyelectrolyte brushes chain space, protein spatial structure and conformation was not affected and while the anionic polyelectrolyte brush has a high adsorption capacity to protein. The pH value of the medium affects the adsorption property greatly. The adsorption capacities of TRY and LZM on CPVA-g-PSSS first increases and then declines with the increase of pH value, and there are maximum adsorption capacities(82mg/g for TRY and 78mg/g for LZM) as the pH value of the medium is equal to their isoelectric points. During the adsorption process, the entropy of the system increases and the heat of the system were released. The adsorption process isdriven by entropy.