Recombinant expression, characterization and expressional analysis of clam Meretrix meretrix cathepsin B, an enzyme involved in nutrient digestion
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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, Vmax and k cat are 6.11 μM, 0.0174 μM 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.
KeywordsCathepsin B Expression and purification Meretrix meretrix Nutrient digestion
We thank Professor Li Sun for her advices and critical reading of the manuscript and also thank the reviewers for their suggestions in revising this work. This work was financially supported by NSFC (30871910), National Basic Research Program of China (2010CB126403) and the National High-Tech R&D Program (2006AA10A410).
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