Characterization and large-scale expression of the recombinant cysteine proteinase from adult Clonorchis sinensis.
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).
Park
S Y
SY
Department of Parasitology, College of Medicine, Wonkwang University, Iksan, Korea.
Lee
K H
KH
Hwang
Y B
YB
Kim
K Y
KY
Park
S K
SK
Hwang
H A
HA
Sakanari
J A
JA
Hong
K M
KM
Kim
S I
SI
Park
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis* / Q000235:genetics; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D017386:Sequence Homology, Amino Acid
D015853:Cysteine Proteinase Inhibitors; D015801:Helminth Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L