Glaxo Institute for Molecular Biology, 14 Chemin des Aulx, CH 1228, Plan les Ouates, Switzerland.
Cytotechnology. 1997 May;24(1):65-72. doi: 10.1023/A:1007974121182.
We report here the successful scale up of transient recombinant protein expression to litre scale using Semliki Forest Virus System. The expression of bacterial β-galactosidase was initially compared in BHK and CHO cells and the conditions for optimal infection of BHK cells were identified. 10% FCS in a medium at pH 6.9 and infection in small volumes were found to be optimal. A high MOI results in an increased recombinant protein yield. Stirring does not affect the infection process. Finally we applied these optimal conditions to the production of a microsomal enzyme, human cyclooxygenase-2 in suspension spinners. Five independant productions at the 1 litre scale yielded reproducible substantial amounts of recombinant protein (16 mg microsomal protein 10(9) cells(-1)) with an average specific activity of 3942 ± 765 pg PGE(2) μg(-1) microsomal protein 5 min(-1).
我们在此报告，使用 Semliki Forest 病毒系统成功地将瞬时重组蛋白表达从微量规模放大到升规模。最初比较了细菌β-半乳糖苷酶在 BHK 和 CHO 细胞中的表达，并确定了 BHK 细胞最佳感染的条件。在 pH 6.9 的培养基中添加 10% FCS 和小体积感染被发现是最佳的。高 MOI 导致重组蛋白产量增加。搅拌不会影响感染过程。最后，我们将这些最佳条件应用于悬浮Spinner 中生产微粒体酶，人环氧化酶-2。5 次独立的 1 升规模生产可重复获得大量的重组蛋白（16mg 微粒体蛋白 10（9）细胞（-1）），平均比活为 3942 ± 765pg PGE（2）μg（-1）微粒体蛋白 5min（-1）。
