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Investigates the role of chaperones in the folding of fusion proteins from Escherichia coli. Production of fusion proteins with predicted molecular masses; Use of luciferases in biotechnology applications; Means of analyzing proteins.

HscC, a DnaK homolog in Escherichia coli, consists of adenosine triphosphatase (ATPase), substrate-binding and C-terminal domains. Overexpression of HscC markedly inhibits growth of host cell and reduces the σ70-dependent promoter activity presumably by forming a complex with σ70. To identify the region(s) of HscC responsible for growth inhibition and complex formation with σ70, domain swapping experiments were carried out between DnaK and HscC. Thus the chimeric proteins carrying the ATPase domain of HscC and substrate-binding domains of either HscC or DnaK were found to inhibit the growth of the cell, reduce the σ70-dependent promoter activity and form a complex with σ70. These results indicate that the ATPase domain of HscC rather than the substrate-binding domain is important for determining its functional specificity. [Copyright &y& Elsevier]

Abstract Background: HscC, the third member of the Hsp70 family in Escherichia coli , shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity. Genetic and biochemical evidence indicates that the DnaK-DnaJ chaperone system interacts with σ32 and is involved in the negative regulation of the heat shock response. Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown. Results: We observed that the over-expression of HscC caused severe growth inhibition. To explore this effect, we performed primer extension analysis and a β-galactosidase assay and found that HscC reduced the σ70 -dependent promoter activity. An in vitro transcription assay revealed that HscC inhibited σ70 -dependent transcription. In addition, the co-purification analysis showed that σ70 co-eluted with HscC. Conclusion: These results indicate that HscC forms a complex with σ70 and may function as its negative modulator. [ABSTRACT FROM AUTHOR]

Abstract Background: HscC, the third member of the Hsp70 family in Escherichia coli , shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity. Genetic and biochemical evidence indicates that the DnaK-DnaJ chaperone system interacts with σ32 and is involved in the negative regulation of the heat shock response. Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown. Results: We observed that the over-expression of HscC caused severe growth inhibition. To explore this effect, we performed primer extension analysis and a β-galactosidase assay and found that HscC reduced the σ70 -dependent promoter activity. An in vitro transcription assay revealed that HscC inhibited σ70 -dependent transcription. In addition, the co-purification analysis showed that σ70 co-eluted with HscC. Conclusion: These results indicate that HscC forms a complex with σ70 and may function as its negative modulator. [ABSTRACT FROM AUTHOR]

Protein--protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology. [ABSTRACT FROM AUTHOR]