Cloning, expression and purification of alkylhydroperoxidase C, a crucial respiratory antioxidant in Brucella abortus

Abstract

During infection,<em> Brucellae</em> face extensive oxidative pressure inside host macrophages in the form of oxidative burst and reactive oxygen species (ROS). Alkylhydroperoxidase C (AhpC) is the primary scavenger of these ROS in <em>Brucella</em> and alkylhydroperoxidase D (AhpD) helps it to regain its reduced state after the catalytic reaction. In this study, we attempted to crystallize the AhpC protein from <em>B. abortus. </em>Extensive crystallization attempts were carried out but crystals could not be obtained. <em>B. abortus</em> <em>AhpC</em> (<em>BaAhpC</em>) gene was cloned and expressed in <em>Escherichia coli</em>, followed by protein purification using affinity and gel filtration chromatography. BaAhpC was found to be a dimer in solution. Protein sequence alignment analysis shows that BaAhpC share about 50% identity with <em>Mycobacterium tuberculosis</em> AhpC (MtbAhpC) protein and has 3 conserved cysteine residues which are directly involved in catalytic activity. The homology model of BaAhpC structure was prepared using MtbAhpC as a template which show it may exist in the form a dodecamer.