The expression, isolation and analysis of Staphylococcus aureus ClpB

Abstract

ClpB is a member of the highly conserved Clp family of heat shock proteins that has been shown to be essential for thermotolerance in bacteria and eukaryotes. Although mutation analysis has been conducted on the S. aureus clpB gene, the protein has not been studied in great detail. Heterologous expression and purification of ClpB, and other heat shock proteins DnaK, DnaJ and GrpE from S. aureus has been performed via the addition of a 6X-histidine tags to allow separation and purification of the staphylococcal proteins from those of E.coli. DnaK, DnaJ and GrpE modulate the activity of ClpB and together, have been found to be essential for the suppression and reversal of protein aggregates in E. coli. Because it has been demonstrated in several prokaryotic systems that the DnaJ/DnaK/GrpE proteins from one species will not support ClpB activity from another, we are examining how these S. aureus proteins modulate the function and biochemical activities of staphylococcal ClpB. As is common to several bacterial clpB genes, staphylococcal ClpB protein expression results in the dual translation of two proteins of differing size (a full length product and one of lower molecular weight), and future experiments will investigate the functional significance of these forms, if any, and their interactions with other heat shock proteins.