Increase in IgG-binding Capacity of Recombinant Protein a Immobilized on Heterofunctional Amino and Epoxy Agarose

摘要

Protein an affinity materials were widely used in purification of monoclonal antibodies and treatment of autoantibody-associated diseases.Due to the low binding capacity of protein A affinity materials compared to other ion exchange materials,protein A affinity materials are often discussed to be the bottleneck among current purification processes and diseases treatment.In this study,the heterofunctional amino and epoxy agarose was used to immobilize the recombinant protein A(rSpA).The immobilization courses of rSpA and the effect of pH and ionic strength and amino density of the heterofunctional supports on the coupling efficiencies of rSpA were investigated.The optimum conditions for rSpA immobilized on the heterofuntional supports were determined as pH 8.0,the ionic strength of 5 mM borate buffer,and the amino density of 15 μmol g-1.Moreover,the 3D structure of B domain from protein A was analyzed by PyMol software and the web-based program MemBrain.The most likely immobilization sites of B domain were located at the Lys-49 and Lys-50,exposing the IgG-binding pocket to the medium.After immobilization,the remaining epoxy groups were blocked with the ethanolamine to obtain the rSpA affinity materials with different ligand density for antibody adsorption from the human plasma.The static and dynamic IgG-binding capacities were determined to be 42 and 32 mg g-1,respectively.The high IgG-binding capacities could be attributed to the oriented attachment of rSpA on the heterofunctional supports,and these results demonstrate that the heterofunctional amino and epoxy agarose is very promising to permit the oriented covalent attachment of rSpA,and that way to prepare affinity materials with high binding capacity.