Cysteine proteinases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based approach of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for Clonorchis sinensis, we overexpressed and characterized the 24-kDa cysteine proteinase from adult worms. First, the partial cysteine proteinase gene from C. sinensis was cloned by performing reverse transcription polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5′ and the 3′ regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends–polymerase chain reaction (RACE-PCR). The cDNA has an open reading frame of 981 bp, and the deduced amino acid sequence shares similarity with the cathepsin L–like cysteine proteinases from Schistosoma mansoni, Paragonimus westermani metacercaria, Fasciola hepatica, and human cathepsin L by 52%, 47%, 34%, and 29%, respectively. The cysteine proteinase was then overexpressed in the yeast Pichia pastoris as an active enzyme on a large-scale basis (19.7 mg/L). The active recombinant enzyme was purified from culture media using a Ni2+–NTA–agarose affinity column and gel filtration chromatography. This 24-kDa recombinant protein exhibited a substrate preference for Z-Phe-Arg-AMC (benzyloxycarbonyl-l-phenylalanyl-l-arginine-7-amino-4-methyl-coumarin) compared with Z-Arg-Arg-AMC, and the activity was inhibited by E-64 (l-trans-epoxysuccinylleucylamido(4-quanidino)butane).
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December 2001
THERAPEUTICS-DIAGNOSTICS|
December 01 2001
CHARACTERIZATION AND LARGE-SCALE EXPRESSION OF THE RECOMBINANT CYSTEINE PROTEINASE FROM ADULT CLONORCHIS SINENSIS
Seong Y. Park;
Seong Y. Park
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
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Kil H. Lee;
Kil H. Lee
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
* Department of Chemical Engineering, National University of Kunsan, Kunsan Chonbuk, 573-360 Korea.
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Young B. Hwang;
Young B. Hwang
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
* Department of Chemical Engineering, National University of Kunsan, Kunsan Chonbuk, 573-360 Korea.
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Kwang Y. Kim;
Kwang Y. Kim
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
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Seung K. Park;
Seung K. Park
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
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H. A. Hwang;
H. A. Hwang
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
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Judy A. Sakanari;
Judy A. Sakanari
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
† Department of Biology, Sonoma State University, Rohnert Park, California 94928.
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Kyeong M. Hong;
Kyeong M. Hong
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
‡ Department of Biochemistry, College of Medicine, WonKwang University, Iksan, 570-749 Korea.
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Suk-il Kim;
Suk-il Kim
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
§ Department of Parasitology, Chosun University College of Medicine, Kwangju 501-759 Korea.
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Hyun Park
Hyun Park
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, 570-749, Korea
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J Parasitol (2001) 87 (6): 1454–1458.
Citation
Seong Y. Park, Kil H. Lee, Young B. Hwang, Kwang Y. Kim, Seung K. Park, H. A. Hwang, Judy A. Sakanari, Kyeong M. Hong, Suk-il Kim, Hyun Park; CHARACTERIZATION AND LARGE-SCALE EXPRESSION OF THE RECOMBINANT CYSTEINE PROTEINASE FROM ADULT CLONORCHIS SINENSIS. J Parasitol 1 December 2001; 87 (6): 1454–1458. doi: https://doi.org/10.1645/0022-3395(2001)087[1454:CALSEO]2.0.CO;2
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