High Expression Of Cathepsin L

Sea cucumber is an important kind of aquatic and economic animal because of itsabundant nutrition and bioactive substance. More attention has been paid to cultivation of seacucumber and its medicinal purposes value. Many kinds of physics and chemical factorswould cause sea cucumber autolysis. This autolytic phenomenon has developed into a majorproblem in fresh sea cucumber preservation, transport and product processing. Studies haveshown that this ability is common to many aquatic organisms and results from the highendogenous proteolytic activity in the body wall of the sea cucumber. So understandingcharacters of cathepsins in sea cucumber will provide an important message to control andutilize the autolysis of sea cucumber.Cathepsin L is the most abundant cysteine protease with only endopeptidase activity. Thecathepsin L is responsible for intracellular protein degradation, and it is also involved in manyother important physiological roles such as antigen presentation, tumor invasion andmetastasis, bone resorption and apoptosis. The cDNA coding for cathepsin L from theintestine of the sea cucumber Stichopus japonicus (GenBank accession number: EU143709)was cloned. The ORF was999bp and the ORF encoded332aa including a signal peptide of16aa at the N-terminus and a mature peptide of316aa. The cathepsin L from S. japonicusshowed significantly sequence homology with the sea urchin, sea anemone and others. Twohighly conserved sequences ERFNIN and GNFD and three catalytic residues Cys139, His278and Asn299were detected in the mutiple sequence alignment. To gain insight into the invitro activities of SjCL, the gene encoding cathepsin L was subcloned into the expressionvector pET32a(+) to construct the recombinant plasmid pET32a(+)-SjCL, and thentransformed into E. coli BL21(DE3) pLysS. After inducing by isopropylthio-β-D-galactoside(IPTG), the recombinant protein was expressed as inclusion bodies. The recombinant proteinwas then denatured, partially purified and refolded to be an active form. The recombinantSjCL protein was shown a higher activity using the synthetic substrate Z-Phe-Arg-Nmec(substrate specific for cathepsin L), however, no activity detected if using another synthetic substrate Z-Arg-Arg-Nmec (substrate specific for cathepsin B). The bioinformatics analysisand expression of cathepsin L from the sea cucumber S. japonicus provide the bases for itsfurther research.