MOLECULAR-CLONING OF THE DNAK LOCUS, AND PURIFICATION AND CHARACTERIZATION OF A DNAK PROTEIN FROM BACILLUS-BREVIS HPD31

Using part of the dnaK gene from Bacillus subtilis as a probe, a 4.4-k bp SacI-Bg/II fragment of chromosomal DNA of Bacillus brevis, a protei n-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues, We p urified DnaK protein to homogeneity from B. brevis HPD31 harboring a m ulticopy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of gluta thione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion pr oteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at abo ut 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25x10(-3) unit/mg protein (the turnover number against ATP was 0 .47 min(-1)) under our assay conditions. The DnaK dimers dissociated i nto monomers on addition of ATP, GTP, CTP, UTP and ATP gamma S, but no t ADP or AMP. DnaK formed a stable complex with permanently unfolded c arboxymethylated alpha-lactalbumin but not with native alpha-lactalbum in, and this complex was dissociated by addition of ATP/Mg, Formation of this complex was inhibited in the presence of inorganic phosphate. (C) 1998 Elsevier Science B.V. All rights reserved.