Synthesis Of Biofunctionalized Nano Chitosan And Their Application In MBP-tagged Protein

Proteins are important carriers of life activities and play an important role in the life of organisms,while protein isolation and purification play a crucial role in proteomics.Nano-chitosan has good biocompatibility and high reactivity ascribed to its surface active amino group,which is why it is considered as a promising biological carrier material.In this study,chitosan nanoparticle was used as the purification vector of maltose binding protein tagged（MBP-tagged）fusion protein,and it was surface-functionalized and used for the separation and purification of the MBP-tagged fusion protein.The main research contents and results of this thesis are as follows:（1）Preparation of fluorescently and biologically active chain-like chitosan nanocomposite and its use in separating MBP-tagged proteins and as fluorescent tracer of tobaccoChain-like chitosan-tripolyphosphate（CS-TPP）nanocomposites were prepared by solution method.Onto the surface of CS-TPP was incorporated 6-carboxyfluorescein（6-FAM）possessing fluorescence activity to obtain CS-TPP-6-FAM nanocomposite.Further,bioactive carboxymethyl-β-cyclodextrin（CβCD）was loaded onto the surface of CS-TPP-6-FAM at a high dosage to obtain CS-TPP-6-FAM-CβCD nanocomposite which can achieve the efficient and rapid purification of MBP-tagged fusion protein.Researches demonstrate that CS-TPP-6-FAM-CβCD nanocomposite has an average diameter of 22 nm and a chain length of 70 nm.Its adsorption for MBP-tagged fusion protein is consistent with Langmuir isotherm equation,belonging to a single molecular layer adsorption.Corresponding adsorption kinetics are in accordance with the quasi-second-order kinetic model,which indicates that the rate-controlling step of the adsorption process is chemical reaction.Besides,CS-TPP-6-FAM-CβCD nanocomposite dispersed in Hoagland nutrient solution can be adsorbed by tobacco through vascular bundle channels;and the fluorescent signals of the nano-materials can be monitored in the roots,stems and leaves of tobacco after 24 h of absorption.Moreover,the as-prepared CS-6-FAM-CβCD nanocomposite has good biocompatibility,while CS-TPP-6-FAM-CβCD nanocomposite has a good application prospect in the efficient purification of MBP-tagged fusion protein from Escherichia coli lysate and as a fluorescent tracer for monitoring plant growth.（2）Preparation of magnetically responsive nano-chitosan for efficient and rapid purification of MBP-tagged fusion proteinFe₃O₄ nanoparticles were prepared by one-step solvothermal method.Biocompatible chitosan was deposited onto the surface of Fe₃O₄ nanoparticles by anionic cross-linking to afford Fe₃O₄@CS nanocomposite.The effects of the mass of chitosan precursor,pH,mass ratio of crosslinking agent to chitosan precursor,and reaction temperature on the morphology of Fe₃O₄@CS nanocomposite were investigated.Furthermore,magnetically responsive Fe₃O₄@CS/Dextrin nanocomposite with a particle size of 570 nm and a chitosan shell thickness of 20 nm was prepared by grafting dextrin onto the surface of Fe₃O₄@CS nanocomposite through hydrogen bonding;and the use of Fe₃O₄@CS/Dextrin nanocomposite for the rapid purification of MBP-tagged proteins from Escherichia coli lysate was investigated.The results show that the as-obtained Fe₃O₄@CS/Dextrin nanocomposite has excellent magnetic response.Its adsorption kinetics for MBP-tagged protein follow Langmuir isotherm equation and quasi-second-order kinetic model.Particularly,it is applicable to the fast and efficient purification of MBP-tagged fusion proteins,while it exhibits desired universality and recycling ability.（3）Construction of a universal magnetically responsive nano-chitosan platform for protein purificationCarboxyl group was grafted onto the surface of magnetically responsive Fe₃O₄@CS nanocomposite by acrylic acid polymerization to obtain Fe₃O₄@CS/PAA composite（PAA refers to polyacrylic acid）.The as-obtained Fe₃O₄@CS/PAA nanocomposite was loaded with different protein antibody ligands including maltose binding protein（MBP）,glutathione S transferase（GST）,and histidine（His）by amidation reaction,thereby achieving the separation and purification of the MBP-tagged,GST-tagged and His-tagged fusion proteins.The results show that the as-prepared Fe₃O₄@CS/PAA nanocomposite has a high binding ability to MBP-tagged,GST-tagged and His-tagged fusion proteins as well as desired universality and recycling ability.In one word,the carboxylated magnetically responsive Fe₃O₄@CS/PAA nanocomposite can be used to construct the platform for the fast,highly efficient,and specific purification of the MBP-tagged,GST-tagged and His-tagged fusion proteins,thereby adding to its value of application.