DnaK is an acidic 70kD Escherichia coli heat shock protein which belongs to the molecular chaperone class of proteins (1). The sequence of the dnaK gene is highly homologous with eukaryotic Hsp70 proteins. Reversible binding of Hsp70 to unfolded nascent polypeptides, partially denatured proteins and also some native proteins leads to: i) the stabilization of polypeptide chains during de novo folding, ii) protection of other proteins from aggregation, iii) reactivation of heat inactivated enzymes, iv) promotion of peptide translocation through membranes, and v) exposure of partially denatured proteins for efficient proteolysis. The N-terminal domains of Hsp70 proteins contain an ATPase active site, and the C-terminal domains contain a peptide binding site. DnaK has been shown to bind to various aberrant proteins including protein fragments, mutant proteins, unfolded proteins as well as native proteins: lambdaP, lambdaO and sigma32. DnaKs ATPase activity is stimulated by aromatic and aliphatic amino acids. The E. coli DnaK protein can be autophosphorylated, and like all Hsp70 members possesses a weak ATPase activity (1). The ADP created during this reaction remains bound to DnaK and can be released by GrpE co-chaperone protein. DnaKs ATPase activity is stimulated up to 50 times by the joint presence of the co-chaperones DnaJ and GrpE heat shock proteins. DnaK changes its conformation in the presence of ATP, triggering release of the protein substrate from the DnaK complex. Many in vitro functions of DnaK protein are potentiated by cooperation with the E. coli molecular chaperones DnaJ and GrpE: 1. Initiation of lambdaDNA replication. DnaK (in the presence of DnaJ and GrpE proteins) partially disaggregates the proprimosomal complex, allowing DnaB helicase to promote unwinding of double stranded DNA near the ori lambdasequence. 2. Protection of other proteins from aggregation and involvement in protein folding. 3. Reactivation of heat-inactivated enzymes: E. coli RNA polymerase, luciferase, DNA polymerase III from E. coli. 4. Activation of native protein for binding to specific DNA sequence: the anti-oncogene p53, P1repA protein, dnaA protein and lambdaO protein. 5. Activation of mutant proteins. 6. Sequestering sigma32 transcription factor from the RNA polymerase core and probably exposing sigma32 for efficient proteolysis. 7. Facilitating protein translocation through intracellular Applications: Suitable for use in Western Blot, ELISA and ATPase activity assay. Other applications not tested Recommended Dilution: Western Blot: dilute into appropriate volume of an SDS containing sample buffer and heated at 100C for 5 minutes prior to use. On a 15-well minigel system, 100ng of protein per lane should be sufficient when used with a colorimetric system. Functional Activity Assay: This protein has tested positive for ATP hydrolysis in an ATPase assay. Optimal dilutions to be determined by the researcher. Storage and Stability: For long-term storage, aliquot to avoid repeated freezing and thawing and freeze at -70C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for 6 months after receipt.
