Preparation Of Protein A Immunoadsorption Materials By Site-controlled Covalent Immobilization

Protein A has high specific binding ability with human immunoglobulin.Protein A is commonly used as ligands to prepare protein A immunoadsorption materials,biochips or immune sensor.Protein A immunoadsorption material can be used as an adsorbent for the treatment of autoimmune diseases.This characteristic of binding human immunoglobulin can be employed to relieve and control the disease through cellular and humoral immunomodulation.Protein A affinity adsorbent with super high antibody-binding capacity plays a prominent part in purification of biopharmaceuticals to decrease the manufacturing costs.We describe a site-specific covalent protein conjugation strategy on agarose beads,at the C terminus of recombinant protein A,by the thiol-maleimide reaction.As comparison,the recombinant protein A was randomly immobilized on the aldehyde-functionalized agarose beads via free amino groups on the protein surface.A different protein densities could be immobilized on the agarose beads by site-specific and random conjugation,respectively.The site-specific conjugation of recombinant protein A on the agarose beads was validated through the assay of free SH groups on the adsorbents using the Ellman's reagent.Analysis of IgG-binding capacity from the human plasma showed the maximal IgG-binding capacities for site-specifically and randomly immobilized adsorbents were about 65.2 mg and 30 mg,respectively,per swollen gram of adsorbent.Remarkably,the super high IgG-binding capacity for site-specifically immobilized adsorbent outperformed the existing commercial protein A Sepharose(ca.50mg/g).The orientation of a protein is crucial for its activity after immobilization,and these results demonstrate that the site-specifically conjugated protein molecule is in a functionally active form to interact with IgG with weak steric hindrance.The proposed approach may be an attractive strategy to synthesis affinity adsorbents with high binding capacity.Multifunctional amino-epoxy resins were used to immobilize recombinant protein A by a new two-step method: Firstly,the amino groups on the resins physically adsorb the recombinant protein A,secondly the recombinant protein A was immobilized on the resins by the interaction of epoxy groups on the resins locating in the vicinity of protein A with the amino groups on protein A.The physical adsorption between amino-epoxy resins and recombinant protein A occurred very rapidly.The conditions of the coupling of the resins with protein A were optimized experimentally.The optimal condition was a low concentration salt solution(5 mM)at pH 8.When the density of amino groups on the resins was 15 ?mol/g and the density of epoxy groups was between 75 ?mol/g and 80 ?mol/g,the coupling efficiency reached 90% under this condition.The static hIgG-binding capacity of the synthesized adsorbent reached 38.9 mg/g.The results showed that the amino-epoxy resins has good application prospect for the preparation of immunoadsorption materials...