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Identification and Intracellular Location of MAGE-3 Gene Product¹

Departments of Surgery and Research, University of Basel, Basel, Switzerland [T. K., E. S-T., C. S., A. J., T. W., F. H., M. H., G. C. S.]; Department of Pathology, University of Basel, Basel, Switzerland [F. G.]; and Dipartimento Biotecnologie H.S. Raffaele, Milan, Italy [G. C.]

The human MAGE-3 gene encodes a melanoma antigenic epitope recognized by specific cytotoxic T lymphocytes, but its gene product has not been identified thus far. We produced a recombinant MAGE-3 gene product by expression cloning of the entire reading frame in the context of a fusion protein characterized by a 10-histidine tail, allowing purification by metal chelation on a nickel Sepharose column. The semipurified product was used to generate MAGE-3-specific monoclonal antibodies. One reagent could identify by immunoblotting the native MAGE-3 gene product as a M_r 48,000 protein in lysates of cell lines showing evidence of MAGE-3 gene expression. No apparent cross-reactivity with recombinant or native MAGE-1 gene product was observed. Immunohistochemistry shows that, closely resembling the MAGE-1 gene product, MAGE-3 is a cytoplasmic protein.

¹ This work was supported by grants from the Swiss Cancer League; the Regional Cancer Leagues of Basel-Stadt, Basel-Land, and St. Gallen-Appenzell; the S. Salvatore (Lugano) and Roche (Bern) Research Foundations; the Karl Mayer Foundation (Liechtenstein); and the Swiss National Fund (Grants 31-36295.92 to G. C. S. and 31-39509.93 to A. J.). G. C. was supported by Grants from Associazione Italiana per la Ricerca sul Cancro (7th project; Italy).

² The first two authors contributed equally to this work.

³ To whom requests for reprints should be addressed, at Surgical Research Laboratory, 20 Hebelstrasse, 4031 Basel, Switzerland.

Received 2/21/95. Accepted 4/20/95.

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