title: Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi.
abstract: Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder.
authors.affiliation: Laboratory of Aquatic Biomedicine & Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Sang Phil
authors.initials: SP
authors.lastname: Shin
authors.affiliation: Laboratory of Aquatic Biomedicine & Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Sang Yoon
authors.initials: SY
authors.lastname: Han
authors.affiliation: Laboratory of Aquatic Biomedicine & Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Jee Eun
authors.initials: JE
authors.lastname: Han
authors.affiliation: Laboratory of Aquatic Biomedicine & Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Jin Woo
authors.initials: JW
authors.lastname: Jun
authors.affiliation: Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea.
authors.forename: Ji Hyung
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Laboratory of Aquatic Biomedicine & Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address: parksec@snu.ac.kr.
authors.forename: Se Chang
authors.initials: SC
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016798:Ciliophora / Q000201:enzymology*; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny
chemical_list: D057056:Cysteine Proteases
keywords: Cathepsin L like protease; Cystein proteases; E. coli expression system; Philasterides dicentrarchi; Site direct mutagenesis