Related Experiment Video
Updated: Jun 11, 2026
Functional Cloning Using a Xenopus Oocyte Expression System
Published on: January 30, 2016
Cloning, expression analysis and enzymatic characterization of cathepsin S from olive flounder (Paralichthys
Na Young Kim1, Sang Jung Ahn, A Ram Lee
1Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, South Korea.
Abstract:
Cathepsin S is a critical protease for the regulation of MHC class II immune responses, and thus is a potential target for developing immunosuppressive drugs in the pathogenesis of degenerative and autoimmune diseases. In this study, we cloned a cDNA encoding for cathepsin S (PoCtS) from the olive flounder, Paralichthys olivaceus. The 1170 bp PoCtS cDNA contained an open reading frame of 1014 bp, which consisted of a 25-residue putative signal peptide, a 96-residue propeptide and the 216-residue mature enzyme. The tissue-specific expression pattern of PoCtS, determined via RT-PCR and real-time PCR analysis, revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however IL-1beta, IL-6, IL-8 and PoCtS expression increased significantly in muscle 6h post-injection of bacterial lipopolysaccharide (LPS). The cDNA encoding proenzyme of PoCtS was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4T-1 vector. Also, the recombinant proPoCtS protein was overexpressed in E. coli BL21(DE3) as a 60 kDa fusion protein. Cathepsin S activity was detected through the cleavage of synthetic fluorogenic peptide substrates, such as Z-Val-Val-Arg-AMC and Z-Phe-Arg-AMC. The optimum pH for the protease activity was determined to be 8. This is the first report that characterized the enzymatic properties and analyzed the expression of piscine cathepsin S.
Insights
Researchers cloned and characterized fish cathepsin S (PoCtS), a protease crucial for immune responses. PoCtS expression increased in flounder muscle after bacterial lipopolysaccharide challenge, suggesting its role in fish immunity.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- Cathepsin S is a key protease regulating MHC class II immune responses.
- It is a potential target for immunosuppressive drugs for autoimmune and degenerative diseases.
Purpose of the Study:
- To clone and characterize the cDNA encoding cathepsin S (PoCtS) from olive flounder (Paralichthys olivaceus).
- To analyze the tissue-specific expression pattern of PoCtS.
- To investigate the enzymatic properties of recombinant PoCtS.
Main Methods:
- cDNA cloning and sequencing
- RT-PCR and real-time PCR for expression analysis
- Bacterial expression of recombinant proPoCtS
- Enzyme activity assays using fluorogenic substrates
Main Results:
- The PoCtS cDNA sequence and its translated protein structure were determined.
- PoCtS exhibited ubiquitous expression in healthy flounder tissues.
- Expression of PoCtS and inflammatory cytokines (IL-1beta, IL-6, IL-8) significantly increased in muscle post-LPS injection.
- Recombinant proPoCtS was successfully expressed and showed protease activity with an optimal pH of 8.
Conclusions:
- This study provides the first characterization of piscine cathepsin S.
- PoCtS plays a role in the immune response of olive flounder, particularly in muscle tissue following bacterial challenge.
- The findings contribute to understanding fish immune mechanisms and potential therapeutic targets.
