Human p53 expressed in baculovirus-infected Sf9 cells displays a two-dimensional isoform pattern identical to wild-type p53 from human cells.
Baculovirus expression of human p53 protein, a nuclear cell cycle regulator, was examined in Sf9 cells and compared to native p53 synthesized in primary human cells. Maximum expression of the recombinant p53 protein occurred 48 h postinfection. De novo synthesis of the protein was evident for only 2 days postinfection; however, in pulse-chase studies, 30% of the synthesized protein remained stable up to 5 days. Seventy-seven percent of immunoprecipitated, [35S]-methionine-labeled, recombinant p53 protein resided in the cytoplasm of Sf9 cells, while 15% localized to the nucleus and 8% was released extracellularly. Separation of modified p53 protein, by charge and molecular weight, was accomplished by two-dimensional PAGE, and the electrophoretic pattern of the recombinant protein was identical to the wild-type protein from primary human mammary epithelial cells, indicating that the posttranslational modifications of the recombinant protein in this system are similar to those in primary human cells. Eleven isoforms focused between pI 5.75 and pI 6.5. The recombinant p53 isoforms were phosphorylated by 32P-labeling. Phosphatase digestion of immunoprecipitated p53 effectively removed phosphorous groups from the recombinant protein, reducing the number of isoforms from 11 to 2, demonstrating that phosphorylation is the major posttranslational event in the recombinant protein.
Patterson
R M
RM
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Services, Research Triangle, North Carolina 27709, USA.
He
C
C
Selkirk
J K
JK
Merrick
B A
BA
D000818:Animals; D001345:Autoradiography; D016367:Baculoviridae; D002460:Cell Line; D015870:Gene Expression; D016158:Genes, p53; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D018411:Spodoptera; D013347:Subcellular Fractions / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000302:isolation & purification
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D016159:Tumor Suppressor Protein p53; D008715:Methionine