Coupled transcription-and-translation in Xenopus oocyte and egg extracts.
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.
Tokmakov
Alexander A
AA
Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Japan. tokmak@gsc.riken.go.jp
Terazawa
Yumiko
Y
Ikeda
Mariko
M
Shirouzu
Mikako
M
Yokoyama
Shigeyuki
S
D000818:Animals; D002457:Cell Extracts / Q000494:pharmacology; D002474:Cell-Free System / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D009865:Oocytes / Q000166:cytology*; D010063:Ovum / Q000166:cytology*; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis; D020871:RNA Stability; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism; D014982:Xenopus laevis / Q000502:physiology*
D002457:Cell Extracts; D011994:Recombinant Proteins; D014764:Viral Proteins; D012313:RNA; D008156:Luciferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases