Coupled transcription-and-translation in Xenopus oocyte and egg extracts

Abstract

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 1 h. 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.

Introduction

Oocytes of the African clawed frog, Xenopus laevis, are commonly used for the functional expression of heterologous genes (reviewed in Richter, 1991, Shiokawa et al., 1997, Miller and Zhou, 2000). Two techniques are widely employed to deliver genes into oocytes—cytoplasmic injection of in vitro synthesized mRNA and nuclear injection of cDNA, encoding the protein of interest. Recently, a novel method has been described for heterologous gene expression in Xenopus oocytes. It is based on the cytoplasmic coinjection of purified T7 RNAP and T7 promoter-driven plasmid DNA (Geib et al., 2001). This approach allows simultaneous gene transcription-and-translation (TnT) in the oocyte cytoplasm. Efficient coupling of T7 RNAP-mediated transcription with the intrinsic oocyte translation machinery was shown to occur in the oocyte cytoplasm (Tokmakov et al., 2006).

On the other hand, cell-free systems of protein synthesis have been developed that offer numerous advantages over cell-based expression (reviewed in Yokoyama, 2003, Spirin, 2004, Katzen et al., 2005). Cell-free gene expression allows direct access to the reaction and control of the reaction conditions without concerns about cell growth and viability. Both prokaryotic and eukaryotic expression systems are available for in vitro protein synthesis. The most established systems are those based on Escherichia coli extracts, wheat germ extracts, and rabbit reticulocyte lysates. They are commercially available from several sources. The reactions in these systems can be programmed with RNA or DNA templates. Coupling of transcription and translation using DNA templates and bacteriophage T7 or SP6 RNA polymerases was shown to provide the most efficient protein production in a cell-free environment (Kigawa and Yokoyama, 1991, Kudlicki et al., 1992).

Active, cell-free protein-synthesizing extracts have been prepared from oocytes and eggs of X. laevis (Patrick et al., 1989). These extracts were used for in vitro translation of various heterologous mRNAs (Matthews and Colman, 1995, Matthews and Colman, 1998). Xenopus egg extracts maintain a high capacity for post-translational modifications, such as glycosylation, phosphorylation, signal sequence cleavage, translocation, and assembly of multimeric proteins (Matthews and Colman, 1998). Usually, the translation efficiency of different mRNAs in the egg extracts parallels that seen in Xenopus oocytes. Still, cell-free protein expression in Xenopus oocyte and egg extracts is rarely used in biological studies, and its applications have not been developed. Also, the possibility of a coupled TnT reaction in this system has not been investigated.

In the present study, using luciferase gene as a reporter, we demonstrate that heterologous protein synthesis in Xenopus oocyte and egg extracts can be programmed with T7 RNAP promoter-driven DNA in the presence of T7 RNAP. However, the kinetics of protein and RNA synthesis in the extracts are quite different from those observed in oocytes, reflecting the intrinsically short lifetime of the cell-free expression system.