Study On The Preparation Of Functional Magnetic Fe₃O₄ Nanoparticles And Their Assisted Lysozyme Refolding

In recent years,more and more attention has been paid to nanoparticles based on artificial molecular chaperones,nano-chaperones and functional magnetic nanoparticles to assist protein refolding.Magnetic Fe₃O₄nano-materials have been widely used in many fields,such as life science,because of their good bio-compatibility,low toxicity,easy preparation and so on.In addition,many small molecular compounds related to biology are not only very easy to obtain,but also can be used to modify functional materials,such as cholesterol and 2-methylpropenyloxyethyl phosphorylcholine?MPC?.Therefore,the purpose of this paper is to design and synthesize functional Fe₃O₄composite nanoparticles,and use them as artificial molecular chaperones for the refolding of denatured lysozyme.This article includes three sections as following:1. Literature reviewProtein folding includes two parts of the related research in vitro and in vivo.Under the guidance of protein folding mechanism in vivo,protein refolding methods in vitro have made great progress,and play an important role in promoting the large-scale production of recombinant protein drugs and the industrialization of related products.In this chapter,we will review the new methods and progress of protein refolding based on artificial molecular chaperones in solution and column,including additives,novel chromatographic stationary phase,magnetic Fe₃O₄nanoparticles,and the research meaning and content of this paper.2. Preparation and characterization of functional magnetic Fe₃O₄nanoparticlesSmall biomolecules,like Cholesterol and MPC,whose molecular structure has hydrophobic and electrostatic interaction groups with proteins,which can be used to modify functional magnetic Fe₃O₄nanoparticles.In this chapter,Fe₃O₄was synthesized by coprecipitation method,and obtained Fe₃O₄@PDA nanoparticles by the self polymerization of dopamine.Then,with Fe₃O₄@PDA as a secondary reaction platform,Cholesterol and MPC molecules were modified to prepare functional magnetic Fe₃O₄composite nanoparticles with hydrophobic and electrostatic groups,respectively.The materials were characterized by FT-IR,TGA,X-ray powder diffraction,X-ray photoelectron spectroscopy,transmission electron microscopy and magnetic measurement system.The results show that Fe₃O₄@PDA@Cholesterol and Fe₃O₄@PDA@MPC composite nanoparticles have good appearance and magnetic response properties.Their particle size is about 17 nm and saturation magnetization are 20.6 and 20.9 emu/g,respectively.3. Renaturation of dnatured lsozyme assisted by functional group magnetic Fe₃O₄nanoparticlesThe results show that the advantage of magnetic nanoparticles for protein refolding is that the modified suitable functional group microparticles can precisely regulate the hydrophobic or electrostatic interaction with the denatured protein molecules,and achieve the effective refolding of denatured protein.In this work,the effect of magnetic Fe₃O₄nanoparticles on denatured lysozyme refolding was studied by dilution method.The effects of the amount of the additive,the p H of the refolding buffer and the concentration of Na Cl on the refolding of non reducing and reducing lysozyme were analyzed by fluorescence,synchronous fluorescence and circular dichroism.The results show that when Fe₃O₄@PDA@Cholesterol is used as additive,when the weight of additive is 1.0 mg,the p H value of the refolding solution is 7.5,and the solution does not contain Na Cl,the better refolding effect can be achieved.When Fe₃O₄@PDA@MPC is used as additive,when the weight of additive is 1.0 mg,the p H value of the refolding solution is 7.5,and the solution contains 1.4 mol/L Na Cl,the better refolding effect can be achieved.The results show that the functional magnetic Fe₃O₄composite nanoparticles have renaturation effect on denatured lysozyme.It will be of great application value if more refolding conditions are selected and further study is carried out.