Expression, purification and characterization of the cancer-germline antigen GAGE12I: A candidate for cancer immunotherapy

GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4+ and CD8+ T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I-His by intracellular expression in yeast (Pichia pastoris) and nickel affinity, ion exchange and gel filtration purification. The identity of the purified protein was confirmed by mass spectrometry. This strategy yielded a total of 48 mg of highly pure (>98%) GAGE12I from 8 L of culture (6 mg/l). Interestingly, gel filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins.