title: Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli.
abstract: The kinetics of the heat-shock response and the formation of inclusion bodies in recombinant Escherichia coli TG1 were studied in glucose-limited high-cell-density cultures in response to temperature-induced production of human basic fibroblast growth factor (hFGF-2), a protein which partially aggregates into inclusion bodies. The maximum synthesis rates of heat-shock proteins were similar to those in a control cultivation with a strain carrying an expression vector without inducible structural gene. However, the maximum of induction for many heat-shock proteins including DnaK, ClpB, and HtpG was reached at least 30 min later when synthesis of hFGF-2 was simultaneously induced by the temperature upshift. During this first production phase, hFGF-2 was exclusively deposited in the insoluble cell fraction. Thereafter, accumulation of soluble hFGF-2 was observed, too, indicating that the recombinant protein needs heat-shock chaperones for proper folding at elevated temperatures. Strong recombinant protein production prolonged the synthesis of the majority of heat-shock proteins (including GroELS, DnaK, ClpB, and HtpG) even in a wildtype dnaK(+) background. In contrast, the synthesis rates of the small heat-shock proteins IbpA and IbpB declined within 1 h to preinduction values in control and hFGF-2 producing cultures. In the producing cultivation, IbpA and IbpB synthesis ceased to an undetectable level when soluble hFGF-2 started to accumulate, whereas the synthesis rates of the other heat-shock proteins including those belonging to the DnaK and GroEL families remained high throughout the entire production phase.
authors: Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
authors: F
authors: F
authors: Hoffmann
authors: U
authors: U
authors: Rinas
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018869:Heat-Shock Response; D002479:Inclusion Bodies; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; C063842:dnaK protein, E coli