Tokmakov Alexander A, Terazawa Yumiko, Ikeda Mariko, Shirouzu Mikako, Yokoyama Shigeyuki
Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Japan.
J Biotechnol. 2006 Oct 1;125(4):557-64. doi: 10.1016/j.jbiotec.2006.03.020. Epub 2006 May 2.
Coinjection of T7 promoter-driven plasmids and T7 RNA polymerase (T7 RNAP) into Xenopus oocytes results in robust protein synthesis, due to simultaneous gene transcription-and-translation (TnT) in the oocyte cytoplasm [Geib, S., Sandoz, G., Carlier, E., V. Cornet, Cheynet-Sauvion, V., De Waard, M., 2001. A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase. Receptors Channels 7, 331-343; Tokmakov, A.A., Matsumoto, E., Shirouzu, M., Yokoyama, S., 2006. Coupled cytoplasmic transcription-and-translation--a method of choice for heterologous gene experession in Xenopus oocytes. J. Biotechnol. 122, 5-15]. In the present study, we demonstrate that the TnT reaction of protein synthesis can be reconstituted in cell-free extracts of Xenopus oocytes and eggs. Similar to the reaction in oocytes, the effective coupling of bacteriophage T7 RNAP-mediated transcription with the eukaryotic translation machinery takes place in the Xenopus oocyte and egg extracts. However, the kinetics of protein and RNA production in the extracts are quite different from those observed in oocytes. Potent RNA synthesis in the extracts starts immediately after the addition of T7 promoter-driven DNA and T7 RNAP and continues for about 30 min, followed by RNA degradation. The protein product is detectable in the extracts in 15 min after the initiation of the TnT reaction. Efficient protein synthesis in the extracts continues for about 1h. The productivity of this expression system can be boosted by the additions of an RNase inhibitor and an ATP-regeneration system, and by extract dilution. Kinetic analyses suggested that extending the lifetime of the extracts would further increase their productivity.
将T7启动子驱动的质粒与T7 RNA聚合酶（T7 RNAP）共注射到非洲爪蟾卵母细胞中，可实现强劲的蛋白质合成，这是由于卵母细胞细胞质中同时发生基因转录与翻译（TnT）[Geib, S., Sandoz, G., Carlier, E., V. Cornet, Cheynet-Sauvion, V., De Waard, M., 2001. 一种基于T7驱动质粒与纯化的T7-RNA聚合酶细胞质共注射的新型非洲爪蟾卵母细胞表达系统。受体通道7, 331 - 343；Tokmakov, A.A., Matsumoto, E., Shirouzu, M., Yokoyama, S., 2006. 耦合细胞质转录与翻译——非洲爪蟾卵母细胞中异源基因表达的首选方法。生物技术杂志122, 5 - 15]。在本研究中，我们证明蛋白质合成的TnT反应可在非洲爪蟾卵母细胞和卵的无细胞提取物中重建。与卵母细胞中的反应类似，噬菌体T7 RNAP介导的转录与真核翻译机制在非洲爪蟾卵母细胞和卵提取物中有效耦合。然而，提取物中蛋白质和RNA产生的动力学与在卵母细胞中观察到的情况有很大不同。在添加T7启动子驱动的DNA和T7 RNAP后，提取物中强大的RNA合成立即开始，并持续约30分钟，随后RNA降解。在TnT反应开始15分钟后，提取物中可检测到蛋白质产物。提取物中高效的蛋白质合成持续约1小时。添加核糖核酸酶抑制剂和ATP再生系统以及稀释提取物可提高该表达系统的产量。动力学分析表明，延长提取物的寿命将进一步提高其产量。
