Preparation and immunogenicity of tag-free recombinant human eppin

Abstract

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, 2mg of eppin without a His 6-tag was isolated from 1l of culture with a purity of >95%. The immunogenicity of the eppin was characterized using male Balb/c mice. © 2011 AJA, SIMM &SJTU. All rights reserved.