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

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 recombi...

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Bibliographic Details
Published in:Parasitology International
Main Authors: 박세창, Shin, Sang Phil, Han, Sang Yoon, Han, Jee Eun, Jun, Jin Woo, Kim, Ji Hyung, Park, Se Chang
Format: Article in Journal/Newspaper
Language:English
Published: Elsevier BV 2018
Subjects:
FLOUNDER PARALICHTHYS-OLIVACEUS
CULTURED OLIVE FLOUNDER
SCUTICOCILIATE PROTEINASES
MOLECULAR-CLONING
URONEMA-MARINUM
IN-VITRO
CILIOPHORA
FISH
PARASITE
TURBOT
Philasterides dicentrarchi
Site direct mutagenesis
Cathepsin L like protease
E. coli expression system
Cystein proteases
Turbot
Online Access:https://hdl.handle.net/10371/91224
https://doi.org/10.1016/j.parint.2013.12.007
Description
Summary: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. OAIID:oai:osos.snu.ac.kr:snu2014-01/102/0000030777/1 SEQ:1 PERF_CD:SNU2014-01 EVAL_ITEM_CD:102 USER_ID:0000030777 ADJUST_YN:N EMP_ID:A076079 DEPT_CD:551 CITE_RATE:2.302 FILENAME:1-s2.0-s1383576913002080-main.pdf DEPT_NM:수의학과 EMAIL:parksec@snu.ac.kr SCOPUS_YN:Y CONFIRM:Y N 1

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