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Applied Biochemistry and Biotechnology

, Volume 162, Issue 7, pp 1858–1871 | Cite as

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

  • Sang Jung Ahn
  • Ji Hea Sung
  • Na Young Kim
  • A Ram Lee
  • Soo Jin Jeon
  • Jung-Soo Lee
  • Joong Kyun Kim
  • Joon Ki Chung
  • Hyung Ho LeeEmail author
Article

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.

Keywords

Cysteine protease Cathepsin L Glutathione S-transferase (GST)-fusion protein Mud loach (Misgurnus mizolepisZymography 

Notes

Acknowledgments

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

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Sang Jung Ahn
    • 1
    • 2
  • Ji Hea Sung
    • 1
  • Na Young Kim
    • 3
  • A Ram Lee
    • 1
  • Soo Jin Jeon
    • 4
  • Jung-Soo Lee
    • 1
  • Joong Kyun Kim
    • 1
  • Joon Ki Chung
    • 3
  • Hyung Ho Lee
    • 1
    Email author
  1. 1.Department of BiotechnologyPukyong National UniversityBusanKorea
  2. 2.Department of EmbryologyCarnegie Institution of WashingtonBaltimoreUSA
  3. 3.Department of Aquatic Life MedicinePukyong National UniversityBusanKorea
  4. 4.Department of PathologyStony Brook University Medical CenterStony BrookUSA

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