Cloning, purification, structural, and functional characterization of methicillin-resistant Staphylococcus aureus (MRSA252) RsbV protein

Drug resistance bacteria produce various proteins to combat xenobiotic, physical, and environmental stresses. These proteins play a pivotal role in bacterial cell survival, and thus are the focus of extensive research as potential targets for anti-bacterial drug discovery. During the current study, the MRSA252 (Methicillin-resistant Staphylococcus aureus) anti-σB factor antagonist (RsbV) protein was expressed to study its structure, and function in vitro. RsbV and anti-σB factor (RsbW) are involved in the transcription of σB factor under stress conditions to help bacterial cell survival. The production of recombinant RsbV protein is important for ligand binding studies, and for new inhibitor identification. In order to have sufficient yield of RsbV protein for structural and functional analysis, the MRSA252 rsbV:pET25b clone was transformed into the Escherichia coli BL21(DE3). Purified RsbV protein was analyzed for structure and functions by using circular dichroism (CD) spectroscopy, and reactive oxygen species (ROS) activity assay, respectively. The CD analysis demonstrated that MRSA252 RsbV has a globular nature with α-helices, and β-strands. It is stable above the ambient temperature. RsbV protein in a cell-based antioxidant assay showed >20-fold more potent inhibition, as compared to drug ibuprofen. The structural element and functional analysis of RsbV protein indicated that RsbV protein is produced in MRSA252 probably as an active regulatory factor under physical, and chemical stresses.