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Molecular Cloning, Expression, and Characterization of Cathepsin L from Mud Loach (Misgurnus mizolepis)

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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.

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Acknowledgments

This research was supported by a project grant (YSG-RE0602) from Yeongnam Sea Grant, Korea.

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Authors and Affiliations

  1. Department of Biotechnology, Pukyong National University, Busan, 608-737, Korea

    Sang Jung Ahn, Ji Hea Sung, A Ram Lee, Jung-Soo Lee, Joong Kyun Kim & Hyung Ho Lee

  2. Department of Embryology, Carnegie Institution of Washington, Baltimore, MD, 21218, USA

    Sang Jung Ahn

  3. Department of Aquatic Life Medicine, Pukyong National University, Busan, 608-737, Korea

    Na Young Kim & Joon Ki Chung

  4. Department of Pathology, Stony Brook University Medical Center, Stony Brook, NY, 11794-8691, USA

    Soo Jin Jeon

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  1. Sang Jung Ahn
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  2. Ji Hea Sung
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Correspondence to Hyung Ho Lee.

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Ahn, S.J., Sung, J.H., Kim, N.Y. et al. Molecular Cloning, Expression, and Characterization of Cathepsin L from Mud Loach (Misgurnus mizolepis). Appl Biochem Biotechnol 162, 1858–1871 (2010). https://doi.org/10.1007/s12010-010-8964-6

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  • DOI: https://doi.org/10.1007/s12010-010-8964-6

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