DnaJ is a basic 41kD Escherichia coli heat shock protein which belongs to the molecular chaperone class of proteins. The E. coli dnaJ gene is located in an operon with dnaK (1). The sequence of the dnaJ gene shares identity with eukaryotic cytosol and endoplasmic reticulum DnaJ homologues. These homologues are known to be involved in i) protein folding, and membrane translocation of newly synthesized proteins, and ii) initiation of translation. The N-terminal domain of the DnaJ protein constitutes the most highly conserved domain and it is used as the signature sequence of DnaJ family members. This so-called “J” domain is important for DnaJ functions, including interaction with DnaK protein. Bacterial DnaJ protein acts synergistically with the bacterial chaperone DnaK (Hsp70 homologue) and the other co-chaperone GrpE in: 1) suppressing eukaryotic and prokaryotic polypeptide aggregation, thus promoting protein folding, 2) facilitating protein translocation through intracellular compartments or protein secretion, 3) reactivating (“repairing”) some partially aggregated enzymes, such as E. coli RNAP and luciferase, 4) sequestering s32 E. coli transcription factor from the RNA polymerase core, and probably exposing it in such a way the s32 is efficiently proteolysed, 5) activating pre priming complex during initiation of lDNA replication, also involved in the initiation of DNA replication of the P1 and F plasmids. In these reactions, DnaJ protein facilitates binding of DnaK to its substrate by a tagging mechanism and/or by accelerating DnaK-catalyzed ATP hydrolysis promoting formation of DnaK-ADP form which possess a higher affinity for some (but not all) protein substrates. Ability to stimulate the ATPase activity of Hsp70 protein by DnaJ protein is also shared by some of its eukaryotic homologues, (i.e. S. cerevisiae YDJ1). Various Hsp70 proteins can perform specialized functions because they interact effectively with only certain DnaJ homologues. Recently published data suggest that DnaJ protein alone possess chaperone activity. It can bind tightly to some denatured proteins such as rhodanese or luciferase and in so doing, prevent their aggregation or misfolding.
Source:This protein was purified from an E. coli strain overexpressing DnaJ.
Functional Activity Assay:The specific activity of DnaJ was estimated to be 4.6x10e5U/mg, where one unit of activity is defined as the incorporation of 1pmol of nucleotides per 1 min. at 30°C into lambdaDNA during in vitro lambdaDNA replication (1). Tested positive for its ability to stimulate DnaKís ATPase activity in the presence of GrpE (2). It has also tested positive for its reactivation in an assay of heat-inactivated firefly luciferase (2). DnaJ protein is assayed for its ability to stimulate DnaK ATPase activity.
Applications:DnaJ has a tendency to aggregate in solution with ionic strength lower than 0.1M KCI. Where lower salt conditions are required, addition of 0.05% Triton X-100 or Brij 58 is recommended. For Western Blot, on a 15-well Biorad minigel system, 50ng of protein per lane should be sufficient. Other applications not tested.
Storage and Stability:For long-term storage, aliquot to avoid repeated freezing and thawing and freeze at -70°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for at least 12 months.
仕様
Size：50ug
Source Antigen：Recombinant, E. coli
Grade：Highly Purified
Purity：≥ 90%; determined by SDS-PAGE analysis.
Form：Supplied as a liquid in 50mM HEPES, pH 7.4, 300mM KCl, 2mM DTT, 10% glycerol.
