Prevention of hCTLA4Ig-Gal Reduction by L-Cystine 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. Degradation of secreted recombinant protein by protease was noticed. In this study, to maintain the quality of hCTLA4Ig-Gal, effects of L-cystine on the prevention of reduction were investigated. L-Cystine was added at 1, 2, and 4 mM in induction medium and the direct inhibition against the reducing component was investigated. In conclusion, reduction of the hCTLA4Ig-Gal could be prevented by optimizing L-cystine addition strategy during induction phase and after purification of target protein in transgenic rice cell cultures.