Prokaryotic Expression And Purification Of Chimeric Protein AG

Protein A immune adsorption column is the most widely used column which having the best adsorption effect in blood purification for the treatment of autoimmune disease currently. But protein A immune adsorbent has some drawbacks in the30years of clinical application, such as the ability of binding human IgG3is weak, so there is no significant effect for the treatment of systemic lupus erythematosus and expansionary myocarditis.In order to overcome the defects of protein A adsorbent, in this paper we combined some length of gene of protein G which has strong binding ability to human IgG3with the protein A gene which binding antibody, accomplished the prokaryotic expression of the chimeric protein AG, and then prepared the fusion protein absorbent which will make up the shortcoming of protein A adsorbent for the weak binding to IgG3, then enhance its effects for the treatment of systemic lupus erythematosus and myocarditis expansionary.In this paper, we used genetic engineering means, chose the E.coli bacteria as expression strain and the plasmid that contains protein A and protein G gene as templates, successfully cloned the1470bp DNA sequence containing the gene which encodes protein A and protein G antibody binding domain, and connected it to the expression vector--pET23a, constituting a restructuring plasmid pET23a-CPAG and then transformed the plasmid to the E.coli BL21(DE3) to express the fusion protein, obtained the optimal strains E.coli BL21(DE3)(pET23a-CPAG) for chimeric protein AG expression. The chimeric protein AG contains only functional areas which can combine with antibodies. The protein’s molecular weight is about54kDa and the experiment proved that the fusion protein is soluble, and has good affinity activity to the human IgG.We used heating precipitation and PEI precipitation as early separation technologies, DEAE weak anion exchange chromatography and IgG-Fc affinity chromatography as pure separation technologies to separate chimeric protein AG. Through the reasonable selection and combination, we finally determined the relatively optimal purification process to separate the fusion protein. The product is analysised by SDS-PAGE and HPLC, the purity is above98%, total recovery rate is60%. MALDI-TOF-MS for measuring the molecular weight is54070.256Da which is consistent with the calculated value.Further we prepared an adsorbent with the ligand of Chimeric Protein AG. Compared with the protein A adsorbent, CPAG adsorbent has the similar adsorption law in adsorbing antibodies, but it has better effect in the reduction of IgG3, and the antibody binding capacity is higher than the protein A adsorbent. The dynamic combined capacity of CPAG adsorbent (ligand density is9.93mg/mL gel) is35.9mg/mL gel for IgG, and the proportion of immobilized chimeric protein AG combining IgG is about1:1.3, the affinity constant of fixed CPAG is3.8×104L/mol for human IgG, and the balance dissociation constants is3.92mg/mL, the biggest theory binding capacity is92.19mg/mLChimeric protein AG can not only overcome the weak binding of protein A with human IgG3, but also can make up the shortage for protein G to combine with other types of immunoglobulin. It is fixed on solid phase matrix surface to be made as antibodies affinity absorbent, and has potential to be used for the blood purification to treat autoimmune diseases such as systemic lupus erythematosus and expansionary myocarditis.