Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

If You Use a Screen Reader

This content is available through Read Online (Free) program, which relies on page scans. Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.

Characterization and Large-Scale Expression of the Recombinant Cysteine Proteinase from Adult Clonorchis sinensis

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 and Hyun Park
The Journal of Parasitology
Vol. 87, No. 6 (Dec., 2001), pp. 1454-1458
DOI: 10.2307/3285318
Stable URL: http://www.jstor.org/stable/3285318
Page Count: 5
  • Read Online (Free)
  • Subscribe ($19.50)
  • Cite this Item
Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Characterization and Large-Scale Expression of the Recombinant Cysteine Proteinase from Adult Clonorchis sinensis
Preview not available

Abstract

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).

Page Thumbnails

  • Thumbnail: Page 
1454
    1454
  • Thumbnail: Page 
1455
    1455
  • Thumbnail: Page 
1456
    1456
  • Thumbnail: Page 
1457
    1457
  • Thumbnail: Page 
1458
    1458