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| Cloning, Expression, Purification, and Characterization of Recombinant GluV8 |
| DUAN Shu-yan1,2, GUO Huai-zu2,3, LI Jing2, ZHENG Juan1,2, ZHAO Zi-ye1,2, ZHANG Da-peng2,3, WANG Hao3 |
1. School of Pharmacy of Liaocheng University, Liaocheng 252000, China;
2. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai 201203, China;
3. Second Military Medical University, Shanghai 200433, China |
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Abstract The glutamyl endopeptidase gene from Staphylococcus aureus was successfully expressed by cloning into the expression vector pGEX-4T-2 and then transformed into E.coil BL21(DE3). Then the recombinant GluV8 was purified by glutathione-Sepharose affinity column. For deamidating activity assay Z-Phe-Leu-Glu-pNA (L-2135) was used as substrate. The reaction result showed that recombinant GluV8 can effectively hydrolyze the alpha-carboxyl of glutamic acid residue, releasing p-nitroanilide with an activity of 1568U/mg. The optimal temperature and the pH is 42℃ and 8.0 respectively. And it remained most of activity at 50℃ and pH9.0. The specificity of recombinant GluV8 by HPLC and LC-MS/MS was identified. These results suggested that the recombinant protein is a relatively specific proteinase that could be effectively utilized for protein identification.
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Received: 27 January 2014
Published: 25 April 2014
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Cite this article:
DUAN Shu-yan, GUO Huai-zu, LI Jing, ZHENG Juan, ZHAO Zi-ye, ZHANG Da-peng, WANG Hao. Cloning, Expression, Purification, and Characterization of Recombinant GluV8. China Biotechnology, 2014, 34(4): 36-40.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20140406 OR https://manu60.magtech.com.cn/biotech/Y2014/V34/I4/36
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