Characterization and purification of human fos protein generated in insect cells with a baculoviral expression vector.
We generated recombinant baculoviruses that contained the human fos gene and that, upon infection of insect cells, synthesized fos protein. The quantity of fos protein produced was at least 10 to 20 times higher than that observed in any mammalian cells reported so far. The fos protein made in insect cells manifested most of the characteristics of mammalian fos protein, which include (i) 55-kilodalton size, (ii) nuclear localization, (iii) phosphoesterification at serine residues, (iv) identical 35S tryptic peptide maps, (v) ability to make heterodimers with the nuclear jun oncoprotein, and (vi) cooperation with the jun protein to bind to a 12-O-tetradecanoyl-phorbol-13-acetate-responsive element. A 100- to 150-fold purification of the fos protein from infected insect cells was achieved in a single step by immunoaffinity chromatography. Availability of authentic fos protein made by baculoviral vectors in insect cells should allow a more rigorous analysis of its biochemical and biological properties.
Tratner
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Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92138-9216.
De Togni
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Sassone-Corsi
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Verma
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006367:HeLa Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D010449:Peptide Mapping; D010710:Phosphates; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification; D016760:Proto-Oncogene Proteins c-fos; D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000302:isolation & purification; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014357:Trypsin
D000596:Amino Acids; D010710:Phosphates; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D014357:Trypsin; D013755:Tetradecanoylphorbol Acetate