MART-1, a melanoma antigen recognized by T cells-1, is a melanocyte lineage-differentiation antigen expressed only in melanocytes and melanoma cells. This protein is recognized by many T-lymphocyte lines that are human leukocyte antigen (HLA)-A2 restricted and melanoma reactive. These observations have culminated in an array of clinical trials of MART-1 immunization using recombinant viruses or MART-1 immunodominant peptides. Polynucleotide immunization is a promising alternative to recombinant viral vaccines that allows delivery of the full-length cDNA encoding all potential peptide epitopes in a vector that is uncompromised by anti-viral immunity. In preparation for a phase I clinical trial of MART-1 polynucleotide immunization in patients with resected melanoma who were at significant risk for recurrence, the authors constructed a plasmid DNA encoding the MART-1 cDNA under transcriptional regulatory control of the cytomegalovirus immediate early promoter-enhancer and partially deleted intron A. This plasmid directs high-level MART-1 expression in transduced myoblasts and maturing myocytes diffusely throughout the cytoplasm. Immunization of mice with this construct by intramuscular injection elicited MART-1-specific immune responses in all animals. Previous trials of MART-1 immunization have been unable to examine the humoral immune response to MART-1 because of a lack of sufficient, highly purified protein. We have produced and purified Escherichia coli recombinant MART-1 protein using a glutathione-S-transferase fusion protein expression system. Protein staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of MART-1 protein at approximately 20 kD; and Western immunoblotting with an anti-MART-1 monoclonal antibody confirmed a doublet at approximately 20 kD. These findings are consistent with previous reports using different expression systems for recombinant MART-1. This protein preparation functioned well in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MART-1 antibody responses in a mouse model; and a panel of healthy donor human sera showed minimal binding to ELISA plates coated with the protein, supporting its utility in monitoring human anti-MART-1 antibody responses. The glutathione-S-transferase fusion method yielded approximately 200 μg MART-1 per 2-L bacterial culture, enough to coat 100 ELISA plates. © 2000 Lippincott Williams & Wilkins, Inc.