Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

Heussler, Volker; Dobbelaere, D A (1994). Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction. Molecular and biochemical parasitology, 64(1), pp. 11-23. Elsevier 10.1016/0166-6851(94)90130-9

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Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

Item Type:	Journal Article (Original Article)
Division/Institute:	08 Faculty of Science > Department of Biology > Institute of Cell Biology 08 Faculty of Science > Other Institutions > Teaching Staff, Faculty of Science 08 Faculty of Science > Department of Biology > Institute of Cell Biology > Malaria
UniBE Contributor:	Heussler, Volker
Subjects:	500 Science > 570 Life sciences; biology 500 Science
ISSN:	0166-6851
Publisher:	Elsevier
Language:	English
Submitter:	Volker Heussler
Date Deposited:	01 Jun 2016 14:24
Last Modified:	05 Dec 2022 14:56
Publisher DOI:	10.1016/0166-6851(94)90130-9
PubMed ID:	8078514
URI:	https://boris.unibe.ch/id/eprint/82267