title: Molecular cloning, expression, and characterization of cathepsin L from mud loach (Misgurnus mizolepis).
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.
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan, Korea.
authors.forename: Sang Jung
authors.initials: SJ
authors.lastname: Ahn
authors.forename: Ji Hea
authors.initials: JH
authors.lastname: Sung
authors.forename: Na Young
authors.initials: NY
authors.lastname: Kim
authors.forename: A Ram
authors.initials: AR
authors.lastname: Lee
authors.forename: Soo Jin
authors.initials: SJ
authors.lastname: Jeon
authors.forename: Jung-Soo
authors.initials: JS
authors.lastname: Lee
authors.forename: Joong Kyun
authors.initials: JK
authors.lastname: Kim
authors.forename: Joon Ki
authors.initials: JK
authors.lastname: Chung
authors.forename: Hyung Ho
authors.initials: HH
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003531:Cypriniformes / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D056890:Eukaryota / Q000145:classification / Q000201:enzymology / Q000235:genetics; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D029941:Fish Proteins; D011994:Recombinant Proteins; D056668:Cathepsin L