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Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi

Cited 7 time in Web of Science Cited 7 time in Scopus
Authors
Shin, Sang Phil; Han, Sang Yoon; Han, Jee Eun; Jun, Jin Woo; Kim, Ji Hyung; Park, Se Chang
Issue Date
2014-04
Publisher
Elsevier Ireland Ltd
Citation
Parasitology International Vol.63 No.2, pp. 359-365
Keywords
농수해양Philasterides dicentrarchiSite direct mutagenesisCathepsin L like proteaseE. coli expression systemCystein proteases
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.
ISSN
1383-5769
Language
English
URI
https://hdl.handle.net/10371/91224
DOI
https://doi.org/10.1016/j.parint.2013.12.007
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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Journal Papers (저널논문_수의학과)
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