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| Prokaryotic Expression,Purification and Activity Identification of Cysteine Hydrolase IdeS |
| XU Jing1, ZHAO Zi-ye2, XU Jin3,4, GUO Huai-zu3,4, ZHENG Juan2, DUAN Shu-yan2, PENG Xiao-yun5, WANG Hao1 |
1. Second Military Medical University, Shanghai 200433, China;
2. School of Pharmacy, Liaocheng University, Liaocheng 252000, China;
3. School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China;
4. State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai 201203, China;
5. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China |
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Abstract ImmunoglobulinG-degrading enzyme of Streptococcus pyogenes (IdeS) is a kind of typical cysteine hydrolase, which cleaves IgG at a specific site in the hinge area and leads to the formation of F(ab)2 and Fc fractions. Because of its unique specificity and hign activity, IdeS could be used in the preparation of subunits, structural analysis and charaterization of IgG. The recombinant GST-IdeS-His6 was efficiently expressed in Escherichia coli by using a two-tag system. And an enteropeptidase cleavage site was inserted at the N-terminal of IdeS for removing the GST-tag easily. The enterokinase site was also added between amino terminal and GST, in order to remove the GST-tag easily. Purified by affinity chromatography, IdeS was identified and analysised by SDS-PAGE, HPLC-SEC and LC-MS.The results suggested that this system was efficient for the production of IdeS. There would be about 25mg protein, with the purity more than 90%, obtained from one liter bacteria liquid. The protease and antibody IgG could be mixed with 1:100 (m/m) ratio, reacted at 37 ℃ for 30 min, then digested completely. The recombinant IdeS could meet the quality requirement of protein structural analysis and could be further utilized effectively in the characterization analysis of antibody drugs. And this enzyme could be also applied in the research of biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.
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Received: 04 August 2014
Published: 25 October 2014
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Cite this article:
XU Jing, ZHAO Zi-ye, XU Jin, GUO Huai-zu, ZHENG Juan, DUAN Shu-yan, PENG Xiao-yun, WANG Hao. Prokaryotic Expression,Purification and Activity Identification of Cysteine Hydrolase IdeS. China Biotechnology, 2014, 34(10): 8-14.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20141002 OR https://manu60.magtech.com.cn/biotech/Y2014/V34/I10/8
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