Purification of anaphase promoting complex/cyclosome from goldfish oocytes

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-specific ubiquitinating system, including anaphase-promoting complex/cyclosome (APC/C) is thought to be responsible for cyclin B destruction. To learn more about the molecular mechanism of cyclin B degradation, a molecular study of the ubiquitinating system in goldfish has been undertaken. For biochemical preparation of APC/C, we first conducted the cloning, sequencing and expression analysis of goldfish, Carassius auratus, cdc27 that encodes a subunit of APC/C from goldfish ovary. The deduced amino acid sequence is highly homologous to cdc27 from other species. Then recombinant goldfish Cdc27CT (C-terminal half of Cdc27) was expressed in Escherichia coli, and an antibody was raised against purified recombinant protein. Polyclonal antiserum cross-reactive with Cdc27 was obtained. By the assay using the antibody, APC/C was purified by column chromatographs.