Human epididymal protease inhibitor (eppin) may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His(6)-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His(6)-tag must be removed. This study describes a method for producing recombinant human eppin without a His(6)-tag. We constructed plasmid pET28a (+)-His(6)-tobacco etch virus (TEV)-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His(6)-TEV-eppin was digested with TEV protease to remove the His(6)-tag and was further purified by NTA-Ni(2+) affinity chromatography. Using this procedure, 2 mg of eppin without a His(6)-tag was isolated from 1 l of culture with a purity of >95%. The immunogenicity of the eppin was characterized using male Balb/c mice.