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Purification and properties of the Escherichia coli dnaK replication protein
Abstract:
The Escherichia coli dnaK+ gene was cloned into the "runaway" plasmid vector pMOB45 resulting in a large overproduction of the dnaK protein. The dnaK protein was purified by following its ability to complement the replication of single-stranded M13 bacteriophage DNA in a reaction system dependent on the presence of the lambda O and P DNA replication proteins. The DNA replication activity of the dnaK protein is also essential for lambda dv DNA replication in vitro, since antibodies against it were shown to inhibit the reaction. Purified dnaK protein preparations possess a weak ATPase activity and an autophosphorylating activity which copurify with its DNA replication activity throughout all purification steps. The dnaK protein is an acidic largely monomeric protein of Mr = 72,000 and 78,400 under denaturing and native conditions, respectively. The amino acid composition and N-terminal amino acid sequence match those predicted from the DNA sequence of the dnaK gene (Bardwell, J.C.A., and Craig, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 848-852).
Insights
Researchers purified the Escherichia coli dnaK protein, essential for DNA replication. This protein exhibits DNA replication, ATPase, and autophosphorylating activities, crucial for M13 and lambda phage DNA replication.
Area of Science:
- Molecular Biology
- Bacteriology
- Biochemistry
Background:
- The dnaK+ gene of Escherichia coli encodes a crucial protein involved in cellular processes.
- Understanding the function of dnaK protein is vital for comprehending DNA replication mechanisms.
Purpose of the Study:
- To clone and overproduce the Escherichia coli dnaK protein.
- To purify and characterize the DNA replication, ATPase, and autophosphorylating activities of the dnaK protein.
- To investigate the role of dnaK protein in M13 and lambda phage DNA replication.
Main Methods:
- Cloning of the Escherichia coli dnaK+ gene into a "runaway" plasmid vector (pMOB45) for overproduction.
- Purification of dnaK protein based on its ability to complement M13 bacteriophage DNA replication.
- In vitro replication assays using M13 and lambda dv DNA, with antibody inhibition to confirm dnaK protein's essential role.
- Biochemical assays to assess ATPase and autophosphorylating activities of the purified protein.
Main Results:
- Successful cloning and large-scale overproduction of the dnaK protein.
- Purified dnaK protein demonstrated essential DNA replication activity for both M13 and lambda dv DNA in vitro.
- Copurification of DNA replication activity with weak ATPase and autophosphorylating activities.
- Characterization of dnaK protein as an acidic, largely monomeric protein with specific molecular weights under denaturing and native conditions.
Conclusions:
- The purified dnaK protein possesses essential DNA replication, ATPase, and autophosphorylating activities.
- dnaK protein plays a critical role in the replication of M13 and lambda phage DNA.
- The biochemical properties of the purified dnaK protein are consistent with its predicted characteristics from the DNA sequence.