Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
| Recombinant expression, characterization and expressional analysis of clam Meretrix meretrix cathepsin B, an enzyme involved in nutrient digestion | |
Yao, Xueliang1,2; Zhang, Jiquan1; Sun, Jinsheng2; Liu, Baozhong1
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| 2011-03-01 | |
| 发表期刊 | MOLECULAR BIOLOGY REPORTS
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| ISSN | 0301-4851 |
| 卷号 | 38期号:3页码:1861-1868 |
| 文章类型 | Article |
| 摘要 | Cathepsin B is one of the most important proteolytic enzymes involved in the nutrient metabolism of clam Meretrix meretrix. The recombinant fusion protein GST-MmeCB (rGST-MmeCB) was obtained at a high level from Escherichia coli and identified using LC-ESI-MS/MS. The GST tag was cleaved from rGST-MmeCB, and the resulting recombinant MmeCB (rMmeCB) was able to degrade the selective substrate carbobenzoxy-l-arginyl-l-arginyl-7-amino-4-trifluoromethylcoumarin (Z-Arg-Arg-AFC) in vitro. The kinetic parameters of the rMmeCB were calculated as follows: K (m), V(max) and k (cat) are 6.11 mu M, 0.0174 mu M min(-1) and 277.57 s(-1), respectively. Rabbit anti-rGST-MmeCB polyclonal antibodies was prepared and used to analyze the tissue distribution of MmeCB protein in M. meretrix. The results showed that the highest level of cathepsin B was found in the digestive gland and moderate levels were found in gill and mantle. Similar expression patterns were found at the mRNA level as detected by real time PCR. Further analysis showed that starvation caused a slight increase in MmeCB protein synthesis in the digestive gland, while refeeding after starvation caused an apparent increase in MmeCB synthesis in digestive gland, gill and mantle. Real time PCR analysis showed that MmeCB mRNA in digestive gland was significantly up-regulated by starvation and returned to normal level after the starved clams were refed. Together, these results indicated that cathepsin B is probably involved in the nutrient digestion of M. meretrix.; Cathepsin B is one of the most important proteolytic enzymes involved in the nutrient metabolism of clam Meretrix meretrix. The recombinant fusion protein GST-MmeCB (rGST-MmeCB) was obtained at a high level from Escherichia coli and identified using LC-ESI-MS/MS. The GST tag was cleaved from rGST-MmeCB, and the resulting recombinant MmeCB (rMmeCB) was able to degrade the selective substrate carbobenzoxy-l-arginyl-l-arginyl-7-amino-4-trifluoromethylcoumarin (Z-Arg-Arg-AFC) in vitro. The kinetic parameters of the rMmeCB were calculated as follows: K (m), V(max) and k (cat) are 6.11 mu M, 0.0174 mu M min(-1) and 277.57 s(-1), respectively. Rabbit anti-rGST-MmeCB polyclonal antibodies was prepared and used to analyze the tissue distribution of MmeCB protein in M. meretrix. The results showed that the highest level of cathepsin B was found in the digestive gland and moderate levels were found in gill and mantle. Similar expression patterns were found at the mRNA level as detected by real time PCR. Further analysis showed that starvation caused a slight increase in MmeCB protein synthesis in the digestive gland, while refeeding after starvation caused an apparent increase in MmeCB synthesis in digestive gland, gill and mantle. Real time PCR analysis showed that MmeCB mRNA in digestive gland was significantly up-regulated by starvation and returned to normal level after the starved clams were refed. Together, these results indicated that cathepsin B is probably involved in the nutrient digestion of M. meretrix. |
| 关键词 | Cathepsin b Expression And Purification Meretrix Meretrix Nutrient Digestion |
| 学科领域 | Biochemistry & Molecular Biology |
| DOI | 10.1007/s11033-010-0303-z |
| URL | 查看原文 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000286941400053 |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/11929 |
| 专题 | 实验海洋生物学重点实验室 |
| 作者单位 | 1.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 2.Tianjin Normal Univ, Tianjin 300387, Peoples R China |
| 第一作者单位 | 中国科学院海洋研究所 |
| 推荐引用方式 GB/T 7714 | Yao, Xueliang,Zhang, Jiquan,Sun, Jinsheng,et al. Recombinant expression, characterization and expressional analysis of clam Meretrix meretrix cathepsin B, an enzyme involved in nutrient digestion[J]. MOLECULAR BIOLOGY REPORTS,2011,38(3):1861-1868. |
| APA | Yao, Xueliang,Zhang, Jiquan,Sun, Jinsheng,&Liu, Baozhong.(2011).Recombinant expression, characterization and expressional analysis of clam Meretrix meretrix cathepsin B, an enzyme involved in nutrient digestion.MOLECULAR BIOLOGY REPORTS,38(3),1861-1868. |
| MLA | Yao, Xueliang,et al."Recombinant expression, characterization and expressional analysis of clam Meretrix meretrix cathepsin B, an enzyme involved in nutrient digestion".MOLECULAR BIOLOGY REPORTS 38.3(2011):1861-1868. |
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