Molecular cloning, tissue distribution and enzymatic characterization of cathepsin X from olive flounder (Paralichthys olivaceus)

作者：Ahn Sang Jung; Kim Na Young; Jeon Soo Jin; Sung Ji Hea; Je Ju Eun; Seo Jung Soo; Kim Moo Sang; Kim Joong Kyun; Chung Joon Ki; Lee Hyung Ho^*

来源：Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 2008, 151(2): 203-212.

DOI：10.1016/j.cbpb.2008.07.001

• cathepsin X
• cysteine protease
• cDNA cloning
• olive flounder
• Paralichthys olivaceus
• polyclonal antibody

In this study, we have cloned a cDNA encoding for cathepsin X (PoCtX) from the olive flounder, Paralichthys olivaceus. The presence of an HIP motif, which is conserved in the unique cathepsin X family, PoCtX, clearly shows its relation to the cathepsin X group, apart from the cathepsin L or B subfamily. The results of RT-PCR and real-time PCR analyses revealed ubiquitous PoCtX expression in normal and LPS-stimulated tissues. The cDNA encoding for the proenzyme of PoCtX (proPoCtX) was expressed in Escherichia coli as a 57 kDa fusion Protein with glutathione S-transferase. Its activity was quantified via the cleavage of the synthetic fluorogenic peptide substrate Z-Phe-Arg-AMC, and the optimal pH for the protease activity was 5. The recombinant proPoCtX was inhibited by antipain and leupeptin. The PoCtX protein from P. olivaceus muscle extracts was purified 9.48-fold via a one-step purification process using a DEAE-Sephagel high Performance liquid chromatography (HPLC) column. Western blotting and ELISA were conducted in order to evaluate the reaction ability and detection-specificity of the anti-proPoCtX polyclonal antibody to native PoCtX and recombinant proPoCtX proteins. Our findings indicate that the P. olivaceus cathepsin X is highly conserved within the cathepsin X subfamily in terms of its amino acid sequence, tissue expression, and biochemical activity.

• 出版日期2008-10