Confirmation of hCTLA4Ig-Gal Reduction in Transgenic Rice Suspension Cell Cultures

초록

Transgenic rice cell cultures (Oryza sativa L.) with human b1,4-galactosyltransferase (hGalT) gene and inducible RAmy3D promoter were used to express recombinant human galactosylated cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig-Gal), a soluble fusion protein formed as a dimer by disulfide bond. Disulfide bond is one of the most important factor for the quality and stability of immunoglobulin-like protein. It is related with binding affinity, biology activity and potency. RAmy3D promoter is strongly induced by sugar starvation and hCTLA4Ig-Gal is secreted into the medium. The obstacles of this system have been the decrease of cell viability and increase of cell lysis by sugar starvation. Various enzymes released by cell lysis could affect the reducing condition in culture medium during the production phase. In this study, effects of disulfide reduction on the production of hCTLA4Ig-Gal were investigated. Non-reduced Western blot analysis with anti-hCTLA4 and anti-Fc as a first antibody at day 8 showed the reduction of hCTLA4Ig-Gal. To identify the correlation between hCTLA4Ig reduction and cell lysis, both non-reduced Western blot and cell viability analysis were performed. To improve the quality of the biopharceutical protein, suppressing the reduction of disulfide bond is necessary. Regulation of hCTLA4Ig-Gal disulfide is going to be studied further in transgenic rice suspension cell cultures.