Prevention of Plant-derived hCTLA4Ig-Gal Reduction by Cupric Sulfate in Transgenic Rice Cell Cultures

초록

Recently, many recombinant therapeutic proteins are produced using transgenic plant cell cultures due to their advantages as a host for the production of therapeutic proteins, such as low production cost, safety from animal pathogens and capacity for post-translational modifications (PTMs). Especially N-linked glycosylation is one of the most important PTMs associated with protein stability, efficacy and half-life in serum. Transgenic rice cell cultures (Oryza sativa L.) with human b1,4-galactosyltransferase (hGalT) gene and inducible RAmy3D promoter were used to express recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin with hGalT (hCTLA4Ig-Gal), a soluble fusion protein formed as a dimer by disulfide bond. RAmy3D promoter was strongly induced by sugar starvation and the hCTLA4Ig-Gal was secreted into the medium. Disulfide bond is one of the most important factors for maintaining product quality. It is related with binding property, biological activity and potency. To maintain the quality of hCTLA4Ig-Gal, effects of cupric sulfate (CuSO4) on the prevention of reduction were investigated. The copper ions are known as a direct enzyme inhibitor as well as an oxidizing agent. CuSO4 was added at 10, 20 and 40 mM after purification, respectively. From the results of SDS-PAGE under non-reducing condition, it was clear that the reduction of hCTLA4Ig-Gal could be prevented by adding optimized concentration of CuSO4 after the purification of target protein.