摘要
Human epididymal protease inhibitor （eppin） may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His6-tag must be removed. This study describes a method for producing recombinant human eppin without a His6-tag. We constructed plasmid pET28a （＋）-His6-tobacco etch virus （TEV）-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His6-TEV-eppin was digested with TEV protease to remove the His6-tag and was further purified by NTA-Ni2＋ affinity chromatography. Using this procedure, 2 mg of eppin without a His6-tag was isolated from 1 I of culture with a purity of 〉95%. The immunogenicity of the eppin was characterized using male Balb/c mice.
Human epididymal protease inhibitor （eppin） may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His6-tag must be removed. This study describes a method for producing recombinant human eppin without a His6-tag. We constructed plasmid pET28a （＋）-His6-tobacco etch virus （TEV）-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His6-TEV-eppin was digested with TEV protease to remove the His6-tag and was further purified by NTA-Ni2＋ affinity chromatography. Using this procedure, 2 mg of eppin without a His6-tag was isolated from 1 I of culture with a purity of 〉95%. The immunogenicity of the eppin was characterized using male Balb/c mice.
基金
ACKNOWLEDGEMENTS The authors are grateful to Dr Zi-Chun Hua （The State Key Laboratory of Pharmaceutical Biotechnology and Department of Biochemistry, College of Life Science, Nanjing University, China） for kindly providing plasmid pET28a （＋） and TEV endoprotease and to Dr Michael G. O＇Rand （Laboratories for Reproductive Biology and Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, USA） for English-language editing. This study was supported by grants from the Key Project of the National Natural Science Foundation of China （No. 30930079） and the Chinese National Nature Science Foundation （No. 81001257）.
