A vector for the synthesis of cRNAs encoding Myc epitope-tagged proteins in Xenopus laevis oocytes.
We describe a plasmid, pNKS2-myc, designed for convenient in-frame fusion of an antibody-specific epitope sequence to the N terminus of a desired cDNA and subsequent synthesis of transcripts that direct the synthesis of the tagged polypeptide in Xenopus laevis (Xl) oocytes. pNKS2-myc contains an SP6 promoter, followed by the translation initiation sequence of the Na,K-pump beta 3 subunit of Xl and the sequence encoding an epitope derived from the human c-myc proto-oncogene product. Appropriate restriction sites allow one to insert virtually any desired cDNA fragment directly behind the epitope-specific sequence and before a long poly(A) tail. After linearization with EcoRI or NotI, polyadenylated cRNA can be synthesized that is efficiently translated in Xl oocytes. The utility of pNKS2-myc is demonstrated by cloning cDNAs coding for Na,K-pump subunits into this vector and injecting the corresponding cRNAs into oocytes. The tagged mouse beta 1 and beta 2 subunit isoforms could be purified from detergent extracts of these cells by immunoprecipitation with a generally available monoclonal antibody (mAb) to the tag, 9E10, as well as with specific mAb that recognize individual beta subunit isoforms. Under native conditions, endogenous and coexpressed exogenous alpha 1 subunits (the catalytic subunit of the Na,K-pump) were co-precipitated, indicating that the N-terminal addition of the decapeptide epitope has no adverse effect on the folding of beta subunits nor on their assembly with alpha subunits. Furthermore, the Myc-specific mAb likewise precipitated a Myc-tagged Na,K-pump alpha 1 subunit together with any of the co-synthesized beta subunits.
Gloor
S
S
Biochemistry II, ETH Center, Zurich, Switzerland.
Pongs
O
O
Schmalzing
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D016259:Genes, myc; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D000090063:Proto-Oncogene Mas; D018075:RNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D014101:Torpedo / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases; D000254:Sodium-Potassium-Exchanging ATPase