Molecular Cloning and Expression of a Caenorhabditis elegans Cathepsin B-Like Protease

• Published in: Biotechnology (Faisalābād, Pakistan) Pakistan), 2009-06, Vol.8 (2), p.242-247
• Main Authors: Miranda-Mi, E., Zamora-Rui, A., Cossio-Bay, R.
• Format: Article
• Language: English
• Subjects: Baculovirus; Caenorhabditis elegans; Nematoda
• ISSN: 1682-296X
• DOI: 10.3923/biotech.2009.242.247

The present study reports the cloning and expression of a Caenorhabditis elegans Cathepsin B-like Protease (CBP), with the objective of obtaining a recombinant enzyme bearing biochemical properties similar to natural CBP reported in the literature for C. elegans and parasitic nematodes. The gene was isolated by PCR from C. elegans cDNA, resulting amplicon was cloned into a baculovirus expression plasmid, insect cells were used for assembly of a recombinant baculovirus containing the C. elegans CBP gene. Thirty five and 45 kDa recombinant proteins were identified from baculovirus infected crude cells containing a His-tag antigenic marker identified by a specific polyclonal antibody in a Western blot assay, both of these recombinant proteins were capable of digesting gelatin in a SDS-PAGE-gelatin assay. Affinity chromatography purified fractions of this recombinant protease, were assayed for peptidase activity against synthetic fluorogenic peptides, including specific cathepsin B substrates and a caspase tetra peptide substrate, maximum cathepsin activity was detected at pH 6.0 for all synthetic substrates and total inhibition was achieved by cystein protease inhibitor E-64 but not by EDTA, pepstatin or PMSF protease inhibitors. Recombinant C. elegans cathepsin B-like protease can be obtained in large amounts from the infected insect cell culture.