Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, Republic of Korea.
J Ind Microbiol Biotechnol. 2013 Jul;40(7):705-13. doi: 10.1007/s10295-013-1273-7. Epub 2013 Apr 26.
For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25 °C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11 g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.
为了有效控制口蹄疫（FMD），需要针对其病原体口蹄疫病毒（FMDV）开发快速诊断系统和疫苗。为了实现这一目标，需要优化 FMDV VP1 蛋白的高效大规模表达，使其具有高溶解性。我们试图在大肠杆菌中大量表达具有高可溶性的血清型 O FMDV VP1 表位。我们鉴定了血清型非依赖性血清型 O FMDV VP1 表位序列，并将其用于构建谷胱甘肽 S-转移酶（GST）融合蛋白。为了高效生产与 GST 融合的 FMDV VP1 表位（VP1e-GST），我们考察了四种大肠杆菌菌株和三种温度条件。在 25°C 的大肠杆菌 BL21(DE3)中，获得了具有最高可溶性的 VP1e-GST 的最佳表达条件。为了进行高水平生产，在 5 升生物反应器中进行了分批补料培养。当细胞在高密度下诱导并提供复杂的补料溶液时，从 2.9 升培养物中获得了约 11 g 的 VP1e-GST。经过纯化后，VP1 表位被用于免疫兔子，我们证实它诱导了免疫反应。
