EXPRESSION, PURIFICATION, AND FUNCTIONAL-CHARACTERIZATION OF ADENOVIRUS-5 AND ADENOVIRUS-12 E1A PROTEINS PRODUCED IN INSECT CELLS

The 12 S and 13 S E1 A cDNAs from both the Adenovinus (Ad) nononcogenic type 5 and the oncogenic type 12 were overexpressed in an insect cell/baculovirus system. Upon infection of Spodoptera frugiperda cells, the production of E1A proteins reached a level of about 15 μg/106 Cells. The E1A proteins are highly soluble and apparently are processed authentically. They are readily recognized by various antibodies and display phosphorylation patterns similar to those of E1 A proteins synthesized in mammalian cells. Single-step immunoaffinity chromatography was used to purify the Ad5 E1A proteins to near homogeneity under nondenaturing conditions. The Ad5 and Ad12 E1 A proteins are able to form complexes with the retinoblastoma susceptibility gene product (Rb) and other cellular proteins. Interestingly, the presence of a cellular extract seems to be a prerequisite for association between highly purified E1A and Rb polypeptides. © 1992.