Premium
Analysis and Characterization of Staphylococcus aureus ClpB and Associated Co‐Chaperone Proteins dnaK, dnaJ and grpE
Author(s) -
Matthews Trey,
Baumann Richard G.,
Domanski Tammy L
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1025-b
Subject(s) - clpb , chaperone (clinical) , biochemistry , biology , escherichia coli , heat shock protein , protein aggregation , intein , chemistry , gene , medicine , rna , pathology , rna splicing
Members of the Clp family of proteins are ubiquitous and induced in response to environmental pressures including oxidative stress and ‘heat shock’. While ClpB has been examined to a large extent in other prokaryotic systems, the function and regulation of this protein in S. aureus has not been examined in great detail. ClpB acts together with other proteins, DnaK, DnaJ, and GrpE, in a chaperone complex which re‐solubilizes protein aggregates during stress conditions. In several prokaryotic systems, the chaperone proteins from one species will not support ClpB activity from another, so we have cloned and sequence verified S. aureus ClpB, as well as the genes for each co‐chaperone proteins, and over‐expressed them as 6X‐His tagged proteins in E. coli. The expression and solubility of each full‐length tagged protein has been examined with SDS‐PAGE gels and verified using Western blotting analysis. Bacterial growth and purification conditions were optimized, and purification of each has been performed using affinity chromatography through a nickel containing columns. 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). To improve over‐expression and solubility conditions for the purification of active clpB protein, we have also cloned this chaperone as an intein‐chitin binding domain containing protein for one‐step affinity purification on chitin columns. Purified ClpB homologs from other species such as E. coli, exhibit ATPase activity and in vitro protein re‐folding functions, and we are currently examining how the purified S. aureus chaperone proteins modulate similar biochemical activities of our purified preparations of staphylococcal ClpB.