Expression and processing of recombinant human terminal transferase in the baculovirus system.
Overproduction of human terminal transferase protein has now been accomplished by cloning the coding sequence of human terminal transferase into a baculovirus, where the expression of terminal transferase is under the control of the polyhedrin protein promoter. Two constructs were made, one producing a protein containing the entire terminal transferase fused to 12 amino acids from the NH2 terminus of the polyhedrin protein, and the other producing 58-kDa human terminal transferase. The terminal transferase levels expressed in cells infected with either recombinant baculovirus are around 10,000 units/10(7) cells at 48 h postinfection, about 200-fold greater than levels expressed in thymus and cultured lymphoblastoid cells. The chimeric polyhedrin/human terminal transferase protein produced in the infected insect cells has a molecular weight of about 60,000 while the nonfused recombinant human terminal transferase is identical in molecular weight to that present in human lymphoblastoid cells. Both forms of recombinant terminal transferase show immunological and enzymatic activity. When infected cells are pulse-labeled with [35S] methionine at 42-45 h postinfection, about 10% of newly synthesized protein is terminal transferase. Both forms of terminal transferase are phosphorylated in recombinant virus-infected cells as demonstrated by pulse-labeling infected cells with 32P-inorganic phosphate and isolation of labeled terminal transferase peptides by immunoprecipitation.
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004253:DNA Nucleotidylexotransferase / Q000096:biosynthesis* / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis*; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D007118:Immunoassay; D007304:Insect Viruses / Q000201:enzymology* / Q000235:genetics; D007313:Insecta / Q000378:metabolism / Q000382:microbiology; D000077183:Occlusion Body Matrix Proteins; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D015678:Viral Structural Proteins
D004274:DNA, Recombinant; D000077183:Occlusion Body Matrix Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D004247:DNA; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase