Cloning of a cDNA encoding Schistosoma mansoni cathepsin C and expression in the yeast Pichia pastoris
Carty, Charlene (2003) Cloning of a cDNA encoding Schistosoma mansoni cathepsin C and expression in the yeast Pichia pastoris. Master of Science thesis, Dublin City University.
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The cDNA encoding the proenzyme of cathepsin C from the parasitic helminth Schistosoma mansoni was amplified from a cDNA library using 5’ and 3’ specific primers containing sites for SnaBI and Avrll digestion. The full-length cathepsin C cDNA was cloned into the Pichia pastoris expression vector, pPIC9K, in frame with the initiation codon of the yeast a-factor signal sequence. Sequencing of the 1.3 kb cDNA-vector construct revealed a 94.5% identity to the S. mansoni cathepsin C sequence in the public database at amino acid level.
A smaller fragment was also amplified from the library and was cloned into the pGEM vector and sequenced to characterise its identity. This fragment proved to be a truncated version of the cathepsin C cDNA.
Prior to transformation of P. pastoris strain GS115, the pPIC9K-cathepsin C construct was linearised with Bglll. Transformants were selected on the basis of resistance to the antibiotic G418. PCR revealed that the cathepsin C cDNA had been successfully integrated into the yeast genome of transformants.
Protein expression was induced by addition of methanol to a final concentration of 1.0-1.5%, once or twice daily for six days. Recombinant protein was detected by SDS-PAGE. Proteins of 58, 55, 47 and 25 kDa were produced but a western blot and ELISA did not detect the presence of the hexahistidine tag fused to the COOH terminus of the recombinant enzyme. Protease assays were performed to determine if the enzyme was active against the fluorogenic cathepsin C substrate H-Gly-Arg-NHMec. Activity proved low in all instances relative to positive controls.
Biochemical characterisation of Fasciola hepatica cathepsin C was undertaken. The enzyme was demonstrated to exhibit similar properties to its S. mansoni counterpart such as enhancement of activity by the reducing agent DTT and halide ions. A peak of activity was seen at pH 5.5 but also at pH 8.5, which proved to be unique to F hepatica cathepsin C.
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