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영어

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), a fusion recombinant protein, was expressed in transgenic rice cell suspension cultures. The expression vector pMYN409 was constructed to express hCTLA4Ig under the control of RAmy3D promoter. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4Ig into the media. The rice-derived hCTLA4Ig could be purified with affinity chromatography using protein A resin. Recombinant hCTLA4Ig has molecular weight of ~50 kDa on SDS-PAGE under
reducing condition. hCTLA4Ig was produced inducibly by sugar starvation in stirred tank bioreactors with various process strategies. Marine, turbine, and hollowed paddle impeller were compared for the cultivation of transgenic rice cells and optimization of culture medium was performed. Two-stage cultivation method was applied to achieve high cell density during cell growth phase and to enhance the production of hCTLA4Ig. Two kinds of media exchange methods (perfusion and direct exchange) were compared in a 5-L stirred tank bioreactor. In the production phase, inducible production of hCTLA4Ig caused cell death because of sugar depletion, and media exchange induced cell lysis by additional shear stress. It was essential to reduce shear stress for increasing productivity because protease activity was increased by cell lysis. High density cultivation was also investigated in the bioreactor. With fed-batch
cultivation, maximum dry cell weight reached 21.2 g/L and maximum hCTLA4Ig production reached 13.4 mg/L which were 2.18-fold and 2.14-fold higher than those of the direct media exchange, respectively. Therefore, we can conclude that the minimization of cell lysis and achievement of high cell density could be necessary to enhance the production of hCTLA4Ig with RAmy3D promoter system. Control of oxygen level was found to be important. In addition, effect of shear was significant. S225