title: 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, 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.
authors: School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123, China.
authors: Jie
authors: J
authors: Zhang
authors: Xin-Liang
authors: XL
authors: Ding
authors: Zeng-Hui
authors: ZH
authors: Bian
authors: Yan-Kai
authors: YK
authors: Xia
authors: Shou-Lin
authors: SL
authors: Wang
authors: Ling
authors: L
authors: Song
authors: Xin-Ru
authors: XR
authors: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003272:Contraceptive Agents, Male; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D053491:Proteinase Inhibitory Proteins, Secretory / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D003272:Contraceptive Agents, Male; D017931:DNA Primers; C431553:Eppin protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins