Recombinant production and characterisation of two Fasciola hepatica cathepsin L proteases
Collins, Peter Raymond (2005) Recombinant production and characterisation of two Fasciola hepatica cathepsin L proteases. PhD thesis, Dublin City University.
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The cysteine proteases of the liver fluke, Fasciola hepatica, are of great importance to the virulence of the organism. Previous work has established the involvement of these enzymes in catabolism, migration through host tissues and defence against host immune attack. They have therefore been identified as important vaccine targets.
In this study, sequence analyses compare the F. hepatica cathepsins LI and L2 with each other and with other cathepsin Ls of F. hepatica and the related parasite, F. gigantica. Groupings of enzymes based on propeptide and active site cleft sequences are established, indicating possible functional diversity in both species. Comparisons with other trematode and mammalian cathepsin Ls were also performed, focusing on a propeptide motif involved in enzyme processing.
Production of the recombinant enzymes was by expression in the Pichia pastoris yeast expression system, providing sufficient quantities of enzyme for characterisation studies. Enzyme activity against fluorogenic substrates and native collagens was compared, demonstrating differences in substrate specificity and therefore possible function between the two enzymes. Processing and activation of the enzymes was also investigated, with inactive mutant enzymes produced for this purpose. Finally, details are given of various collaborations with other laboratories utilising the recombinant enzymes produced during this project.
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