Expression and Purification of MsaB Protein in Staphylococcus aureus
Marjorie Lam1, Gyan S. Sahukhal2, Mohamed O. Elasri 2
1Mississippi INBRE Research Scholar, Department of Sciences, Mississippi Gulf Coast Community College, Gulfport, MS
2Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS
The msaABCR operon is a four gene operon that plays an important role in biofilm development, controlled cell death, antibiotic resistance, and persister cell formation. MsaB is the only protein produced from this operon; it functions as a DNA-binding protein that can directly regulate other genes based on nutrient availability. We have shown that MsaB binds to the promoter of the capsule operon and to the protease genes, functioning as a transcriptional activator and repressor respectively. Other studies have shown that MsaB is the RNA chaperone. In this study, we cloned the MsaB protein with 6X-his tags either at 3’ end or 5’ ends in pCN51 cadmium inducible vector. The MsaB expression construct was cloned in USA300 LAC msaABCR deletion mutant. Conditions were optimized for the MsaB expression using different concentrations of cadmium chloride before purifying the protein using Ni-NTA column. We then determined the protein size and purity using SDS-PAGE and confirmed using western blot with anti-his antibody. Lastly, we confirmed the protein activity by electrophoretic mobility shift assay (EMSA) with biotin labelled capsule operon promoter. Our future aim is to use homogenous time-resolved fluorescence (HTRF) to determine the binding constant (Kd) of MsaB to the promoter regions of the cap operon and protease. Furthermore, we will measure the affinity of MsaB protein to their targets in the presence of GTP, ATP, AMP, Valine, Leucine, and Isoleucine. At the completion of these experiments, we expect to develop a model for the mechanism of regulation of cap and proteases by MsaB protein in response to nutrient availability.