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

Cited 6 time in WEB OF SCIENCE Cited 7 time in Scopus
Title
Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi
Author(s)
Shin, Sang Phil; Han, Sang Yoon; Han, Jee Eun; Jun, Jin Woo; Kim, Ji Hyung; Park, Se Chang
Publication Year
2014-04
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. (c) 2013 Elsevier Ireland Ltd. All rights reserved.
ISSN
1383-5769
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/2886
DOI
10.1016/j.parint.2013.12.007
Bibliographic Citation
PARASITOLOGY INTERNATIONAL, v.63, no.2, pp.359 - 365, 2014
Publisher
ELSEVIER IRELAND LTD
Subject
FLOUNDER PARALICHTHYS-OLIVACEUS; CULTURED OLIVE FLOUNDER; SCUTICOCILIATE PROTEINASES; MOLECULAR-CLONING; URONEMA-MARINUM; IN-VITRO; CILIOPHORA; FISH; PARASITE; TURBOT
Keywords
Philasterides dicentrarchi; Site direct mutagenesis; Cathepsin L like protease; E. coli expression system; Cystein proteases
Type
Article
Language
English
Document Type
Article
Publisher
ELSEVIER IRELAND LTD
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