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Updated: Jun 13, 2026
High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies
Published on: September 29, 2023
Molecular cloning, expression, and characterization of cathepsin L from mud loach (Misgurnus mizolepis)
Sang Jung Ahn1, Ji Hea Sung, Na Young Kim
1Department of Biotechnology, Pukyong National University, Busan, Korea.
Abstract:
Cathepsin L is an important protease in the initiation of protein degradation and one of the most powerful endopeptidases. In this study, we cloned mud loach (Misgurnus mizolepis) cathepsin L (MlCtL) cDNA, and the pro-mature enzyme of MlCtL (proMlCtL) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4 T-1 vector. The recombinant proMlCtL was overexpressed in E. coli DH5αMCR as a 62-kDa protein. Its activity was quantified by measuring the cleavage of synthetic fluorogenic peptide substrates, and the protease activity of proMlCtL was also demonstrated by gelatin zymography. Antipain and leupeptin were shown to inhibit the protease activity of proMlCtL. Our results suggest that the structural features and evolutionary relationship of the mud loach cathepsin L gene were similar to that of the other mammalian cathepsin Ls; however, the proMlCtL protein was more stable at neutral and alkaline pH. The optimum temperature for the proMlCtL enzyme was found to be 40 °C. In addition, proMlCtL activity was dependent upon the presence of several metal ions and detergents.
Insights
Researchers cloned mud loach cathepsin L (MlCtL) and expressed its pro-mature enzyme in E. coli. The study characterized MlCtL
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cathepsin L is a key protease involved in protein degradation.
- Understanding fish cathepsin L provides insights into evolutionary relationships and enzyme function.
Purpose of the Study:
- To clone and express mud loach cathepsin L (MlCtL) cDNA.
- To characterize the biochemical properties of the recombinant pro-mature MlCtL enzyme.
Main Methods:
- Cloning of MlCtL cDNA and expression in E. coli as a GST-fusion protein.
- Quantification of enzyme activity using fluorogenic peptide substrates.
- Gelatin zymography to confirm protease activity.
- Inhibition assays with antipain and leupeptin.
Main Results:
- Recombinant proMlCtL was successfully overexpressed as a 62-kDa protein.
- Enzyme activity was confirmed and inhibited by antipain and leupeptin.
- ProMlCtL exhibited stability at neutral and alkaline pH, with an optimal temperature of 40 °C.
- Enzyme activity was dependent on metal ions and detergents.
Conclusions:
- Mud loach cathepsin L shares structural and evolutionary similarities with mammalian cathepsin Ls.
- ProMlCtL demonstrates unique stability characteristics at different pH levels.
- The characterization of proMlCtL provides valuable data for understanding fish protease functions.
