Ligand Immobilization Of Protein A Chromatographyand Spectroscopic Investigation Of Structural Transition Of Protein A Ligands

Ligand immobilization techniques are critical for the development of high adsorption capacity protein A of affinity chromatography materials.In order to reveal the interaction mechanism between ligand immobilization technology and immobilized protein A ligand molecule and material interface,this paper uses carboxylated dextran（CMD）coated magnetic nanoparticles（Fe₃O₄@CMD NP）to couple protein A mutant antibody.Protein A nanoparticles were synthesized by binding domain Z（Z domain）and its four-stranded body.On this basis,the interaction between ligand and material interface was systematically observed,which laid a foundation for the development of protein A affinity chromatography media.Magnetic nanoparticles（Fe₃O₄ NP）were synthesized by chemical co-precipitation method,and then CMD was coated on the surface of Fe₃O₄ NP by embedding method,then Fe₃O₄@CMD NP was obtained by SEM,TEM,FT-IR,VSM and TGA.A series of characterization methods indicate that CMD has been successfully wrapped on the surface of Fe₃O₄ NP.The genetically engineered method was used to synthesize the mutant strain of protein A,and then fermented and expressed.Then the affinity product was used to separate and purify the expressed product,and finally the mutant antibody binding domain of protein A was obtained Z-CK and H₆-Z₄-CK.Protein A was immobilized on the surface of Fe₃O₄@CMD NP by orientation fixation and random fixation.In this paper,two protein A ligands were immobilized on the surface of Fe₃O₄@CMD NP by orientation fixation and random fixation.On this basis,the circular dichroism spectroscopy（CD）and fluorescence spectroscopy（FL）techniques were combined with isothermal titration calorimetry（ITC）technology to systematically study the molecular structure and conversion behavior of the protein A ligand on the surface of Fe₃O₄@CMD NP.The results of CD and FL spectroscopy indicated that the molecular structure of the surface ligands of the oriented Z-CK nanoparticles（Fe₃O₄@CMD-o Z NP）was affected by the interaction with the material interface.Under the conditions of p H 4.5 and p H 7.0,the surface of the nanoparticles has a negative charge,which interacts with the side chain of the Lys and Arg residues in the ligand molecule Z-CK to induce conformational changes of the surface ligands of Fe₃O₄@CMD-o Z NP.And the destruction of the structure,whereby theα-helix content of the immobilized ligand molecule is lowered,and the Tyr residue in the ligand molecule is exposed to a hydrophilic environment.The ITC results further show that the combination of Fe₃O₄@CMD-o Z NP with Ig G has a lower stoichiometric coefficient at p H 7.0.When the p H of the buffer is raised to 10.0,the p H of the buffer is close to the p Ka value of the side chain of the Lys residue,and the immobilized ligand has a stable molecular structure on the surface of the Fe₃O₄@CMD NP.Under these conditions,Ig G combination has a higher used.Furthermore,ligand polymerization promoted the structural stability of the ligand by decreasing interaction between NP surface and three domains at amino terminus of the tetrameric ligand.As a result,each tetrameric ligand could bind more Ig G molecule on NP at p H 7.0,the maximum stoichiometric ratio of h Ig G to Fe₃O₄@CMD-o Z NP at p H 10.0 was 1.96.Compared with random immobilization via amino coupling,moreover,oriented immobilization was more benefit for structural stability of the ligand.Spectral and calorimetric evidences at different ligand densities further demonstrated that the crowding effect induced at high ligand density maintained molecular structure of tetrameric ligand better.This research provided an important insight into transition in molecular structure of protein A ligands on chromatographic material for the development of high-capacity protein A chromatography.