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Volume 13, Issue 6 (November 2011) 13, 889–894; 10.1038/aja.2011.89
Preparation and immunogenicity of tag-free recombinant human eppin
Jie Zhang1,*, Xin-Liang Ding2,*, Zeng-Hui Bian3, Yan-Kai Xia4, Shou-Lin Wang4, Ling Song4 and Xin-Ru Wang4
1 School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123, China
2 Department of Public Health, Wuxi Center for Disease Prevention and Control, Wuxi 214023, China
3 Municipal Center for Disease Control and Prevention, Nanjing 210003, China
4 Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 201129, China
* These authors contributed equally to this work.
Correspondence: Dr XR Wang, (xrwang@njmu.edu.cn)
Received 8 November 2010; Revised 7 January 2011; Accepted 21 April 2011; Published online 5 September 2011
| Abstract |
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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.
Keywords: Eppin; immunogenicity; male contraception; recombinant protein preparation; tag-free
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