Novel vectors for expression of cDNA encoding epitope-tagged proteins in mammalian cells.
Two composite constructs, pNHA and pCHA, are described. These plasmids are designed for the expression of cDNA in mammalian cells to produce proteins which are tagged with the hemagglutinin epitope sequence, YPYDVPDYA (HA1). The insertion of cDNA into the multiple cloning sites of these vectors has the advantage of 'tagging' the produced protein at either the N terminus or C terminus. To demonstrate the utility of these vectors, pNHA, containing a full-length cDNA (pNHRP33), was expressed in HeLa cells to produce a HA1-tagged peptide. The resulting peptide was purified from the whole-cell extracts by immunoprecipitation with an antibody to the tag (mAb12CA5). The mRNA was transcribed from a T7 promoter of the pNHRP33 construct, translated in a rabbit reticulocyte assay, and the protein product was purified using mAb12CA5 for the HA1 epitope. Among other possibilities, these vectors can be used to: (1) study protein-protein interactions in a mammalian transcription unit, (2) co-purify associated transcription factors, and (3) purify produced proteins when antibodies are not available.
Pati
U K
UK
Department of Surgery, Yale University School of Medicine, New Haven, CT 06510.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA; D004274:DNA, Recombinant; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors; D006367:HeLa Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000276:immunology; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012319:RNA Polymerase II / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014158:Transcription, Genetic; D014162:Transfection
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D011994:Recombinant Proteins; D004247:DNA; D012319:RNA Polymerase II