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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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
id ftseoulnuniv:oai:s-space.snu.ac.kr:10371/91224
record_format openpolar
spelling ftseoulnuniv:oai:s-space.snu.ac.kr:10371/91224 2023-06-18T03:43:23+02:00 Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi 박세창 Shin, Sang Phil Han, Sang Yoon Han, Jee Eun Jun, Jin Woo Kim, Ji Hyung Park, Se Chang Park, Se Chang 2018-06-22 https://hdl.handle.net/10371/91224 https://doi.org/10.1016/j.parint.2013.12.007 영어 en eng Elsevier BV Parasitology International, Vol.63 No.2, pp.359-365 1383-5769 https://hdl.handle.net/10371/91224 doi:10.1016/j.parint.2013.12.007 000333549600016 2-s2.0-84891648209 37702 2014-01/102/0000030777/1 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 Article ART 2018 ftseoulnuniv https://doi.org/10.1016/j.parint.2013.12.007 2023-06-02T00:36:19Z 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 Article in Journal/Newspaper Turbot Seoul National University: S-Space Parasitology International 63 2 359 365
institution Open Polar
collection Seoul National University: S-Space
op_collection_id ftseoulnuniv
language English
topic 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
spellingShingle 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
박세창
Shin, Sang Phil
Han, Sang Yoon
Han, Jee Eun
Jun, Jin Woo
Kim, Ji Hyung
Park, Se Chang
Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi
topic_facet 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
description 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
author2 Park, Se Chang
format Article in Journal/Newspaper
author 박세창
Shin, Sang Phil
Han, Sang Yoon
Han, Jee Eun
Jun, Jin Woo
Kim, Ji Hyung
Park, Se Chang
author_facet 박세창
Shin, Sang Phil
Han, Sang Yoon
Han, Jee Eun
Jun, Jin Woo
Kim, Ji Hyung
Park, Se Chang
author_sort 박세창
title Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi
title_short Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi
title_full Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi
title_fullStr Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi
title_full_unstemmed Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi
title_sort expression and characterization of cathepsin l-like cysteine protease from philasterides dicentrarchi
publisher Elsevier BV
publishDate 2018
url https://hdl.handle.net/10371/91224
https://doi.org/10.1016/j.parint.2013.12.007
genre Turbot
genre_facet Turbot
op_relation Parasitology International, Vol.63 No.2, pp.359-365
1383-5769
https://hdl.handle.net/10371/91224
doi:10.1016/j.parint.2013.12.007
000333549600016
2-s2.0-84891648209
37702
2014-01/102/0000030777/1
op_doi https://doi.org/10.1016/j.parint.2013.12.007
container_title Parasitology International
container_volume 63
container_issue 2
container_start_page 359
op_container_end_page 365
_version_ 1769009752355897344

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