Evaluation of Gelatinolytic and Collagenolytic Activity of Fasciola hepatica Recombinant Cathepsin-L1.
Cathepsin L1
Collagen
Fasciola hepatica
Gelatin
Recombinant enzyme
Journal
Iranian journal of biotechnology
ISSN: 1728-3043
Titre abrégé: Iran J Biotechnol
Pays: Iran
ID NLM: 101276796
Informations de publication
Date de publication:
Jan 2020
Jan 2020
Historique:
entrez:
5
9
2020
pubmed:
5
9
2020
medline:
5
9
2020
Statut:
epublish
Résumé
Cysteine proteases of the liver fluke, Fasciola hepatica, participate in catabolism of proteins, migration of the fluke through host tissues and combat host immune system. In this study, we evaluated proteolytic activity of F. hepatica recombinant cathepsin L1 (rCL1) against gelatin and collagen as common substrates. The coding sequences of F. hepatica CL1 were cloned and expressed in E. coli, in our previous study. The rCL1 was purified by nickel affinity chromatography with a HisTrap Column. The protein concentrations of the purified fractions were determined by Bradford assay. Rat collagen type-1 was treated with distinct amounts of rCL1 at 37 °C, overnight, and the byproduct was analyzed by SDS-PAGE. Furthermore, we used bovine skin gelatin as zymography substrate to evaluate the gelatinolytic activity of the purified rCL1. Recombinant CL1 was capable to digest intact type-1 collagen within 24 h and the gelatinlytic activity of rCL1 was visible at approximately 37 kDa region, with optimal activity at acidified conditions (pH 4). Findings provide a possible mechanism by which a major secretory molecule of F. hepatica could be involved in parasite survival as well as its pathogenesis.
Sections du résumé
BACKGROUND
BACKGROUND
Cysteine proteases of the liver fluke, Fasciola hepatica, participate in catabolism of proteins, migration of the fluke through host tissues and combat host immune system.
OBJECTIVES
OBJECTIVE
In this study, we evaluated proteolytic activity of F. hepatica recombinant cathepsin L1 (rCL1) against gelatin and collagen as common substrates.
MATERIAL AND METHODS
METHODS
The coding sequences of F. hepatica CL1 were cloned and expressed in E. coli, in our previous study. The rCL1 was purified by nickel affinity chromatography with a HisTrap Column. The protein concentrations of the purified fractions were determined by Bradford assay. Rat collagen type-1 was treated with distinct amounts of rCL1 at 37 °C, overnight, and the byproduct was analyzed by SDS-PAGE. Furthermore, we used bovine skin gelatin as zymography substrate to evaluate the gelatinolytic activity of the purified rCL1.
RESULTS
RESULTS
Recombinant CL1 was capable to digest intact type-1 collagen within 24 h and the gelatinlytic activity of rCL1 was visible at approximately 37 kDa region, with optimal activity at acidified conditions (pH 4).
CONCLUSION
CONCLUSIONS
Findings provide a possible mechanism by which a major secretory molecule of F. hepatica could be involved in parasite survival as well as its pathogenesis.
Identifiants
pubmed: 32884958
doi: 10.30498/IJB.2020.143160.2357
pii: IJB-18-1
pmc: PMC7461709
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e2357Informations de copyright
Copyright: © 2019 The Author(s); Published by Iranian Journal of Biotechnology.
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