Molecular Cloning and Expression of Giardia Cathepsin B

Tapas Haldar, Sandipan Ganguly

Abstract: Giardia lamblia, a protozoan parasite, is a leading cause of waterborne diarrheal disease globally. Cysteine proteases (CPs), particularly cathepsin B-like enzymes, have been implicated in Giardia's pathogenicity, yet their specific roles remain underexplored. The study highlights on the cloning, expression, and functional characterization of CP14019, a cathepsin B-like CP. The CP14019 gene is successfully amplified, cloned into the pET28a vector, and expressed in Escherichia coli BL21 cells. The recombinant protein, predominantly found in the insoluble fraction, was purified via Ni??-affinity chromatography. Western blot analysis confirmed the antibody's specificity, detecting a ~28kDa band corresponding to CP14019 in both recombinant samples and Giardia trophozoite lysates. Functional assays demonstrated that CP14019 exhibits proteolytic activity characteristic of cathepsin B enzymes, effectively degrading tight junction proteins and chemokines such as IL-8, thereby compromising epithelial barrier integrity and modulating host immune responses. These findings underscore CP14019's role in Giardia's virulence and highlight its potential as a target for therapeutic intervention. The generated antibodies provide valuable tools for further investigations into CP14019's function during Giardia infections.

Keywords: Giardia, Diarrhoea, Cathepsin B, Cloning, Protein expression