Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31

Abstract Using part of the dnaK gene from Bacillus subtilis as a probe, a 4.4-kbp Sac I- Bgl II fragment of chromosomal DNA of Bacillus brevis , a protein-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues. We purified DnaK protein to homogeneity from B. brevis HPD31 harboring a multi-copy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S -transferase-DnaJ and glutathione S -transferase-GrpE fusion proteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25×10 −3 unit/mg protein (the turnover number against ATP was 0.47 min −1 ) under our assay conditions. The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPγS, but not ADP or AMP. DnaK formed a stable complex with permanently unfolded carboxymethylated α-lactalbumin but not with native α-lactalbumin, and this complex was dissociated by addition of ATP/Mg. Formation of this complex was inhibited in the presence of inorganic phosphate.