pXen, a utility vector for the expression of GST-fusion proteins in Xenopus laevis oocytes and embryos.
We describe a plasmid, pXen, designed for the optimized expression of proteins fused to glutathione-S-transferase (GST) in Xenopus laevis oocytes and embryos. The Xenopus model system permits the biochemical analysis of signaling pathways and analysis of embryo phenotype in response to manipulation of proto-oncogene expression. pXen is a modified pSP64T vector which contains an SP6 RNA polymerase promoter followed by the translational initiation sequence of Xenopus beta-globin and the glutathione binding domain of GST. The Xenopus 3' beta-globin untranslated region and polyadenylation site immediately follow the multiple cloning site to permit the efficient translation of in vitro transcribed RNA in oocytes and embryos. The utility of pXen is demonstrated by cloning the catalytic domain of the serine/threonine kinase proto-oncogene Raf-1 into this vector and injecting the corresponding in vitro transcribed RNA into oocytes. Catalytically active GST-vRaf fusion protein was expressed in the injected oocytes and induced oocyte maturation. Moreover, the GST-vRaf fusion protein could be readily purified from Xenopus extracts using glutathione Sepharose. We demonstrate that the Raf-1 catalytic domain retains activity when fused with the N-terminal GST moiety and is subject to negative regulation by the cyclic AMP-dependent protein kinase (PKA). The pXen vector will be useful for an in vivo analysis of the physiological role and regulation of a wide variety of signaling molecules when expressed in Xenopus oocytes and embryos.
MacNicol
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Department of Medicine MC6088, University of Chicago, IL 60637, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000502:physiology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D027384:Oncogene Proteins v-raf; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D012313:RNA; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014158:Transcription, Genetic; D014982:Xenopus laevis / Q000196:embryology*
D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D012313:RNA; D005982:Glutathione Transferase; D027384:Oncogene Proteins v-raf; D017868:Cyclic AMP-Dependent Protein Kinases