HUMAN P53 EXPRESSED IN BACULOVIRUS-INFECTED SF9 CELLS DISPLAYS A 2-DIMENSIONAL ISOFORM PATTERN IDENTICAL TO WILD-TYPE P53 FROM HUMAN-CELLS

Baculovirus expression of human p53 protein, a nuclear cell cycle regu lator, was examined in Sf9 cells and compared to native p53 synthesize d in primary human cells, Maximum expression of the recombinant p53 pr otein occurred 48 h postinfection. De novo synthesis of the protein wa s evident for only 2 days postinfection; however, in pulse-chase studi es, 30% of the synthesized protein remained stable up to 5 days. Seven ty-seven percent of immunoprecipitated, [S-35]-methionine-labeled, rec ombinant p53 protein resided in the cytoplasm of Sf9 cells, while 15% localized to the nucleus and 8% was released extracellularly. Separati on of modified p53 protein, by charge and molecular weight, was accomp lished by two-dimensional PAGE, and the electrophoretic pattern of the recombinant protein was identical to the wild-type protein from prima ry human mammary epithelial cells, indicating that the posttranslation al 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 P- 32-labeling, Phosphatase digestion of immunoprecipitated p53 effective ly removed phosphorous groups from the recombinant protein, reducing t he number of isoforms from 11 to 2, demonstrating that phosphorylation is the major posttranslational event in the recombinant protein. (C) 1996 Academic Press, Inc.