On-line estimation of the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins by monitoring respiratory energy generation.
Human basic fibroblast growth factor (hFGF-2) was produced in high-cell density cultures of recombinant Escherichia coli using a temperature-inducible expression system. The synthesis rates of proteins were followed by two-dimensional gel electrophoresis of the (35)S-methionine-labeled proteom. After temperature induction of hFGF-2 synthesis, the rate of total protein synthesis per biomass increased by a factor of three, mainly as a result of the additional synthesis of hFGF-2 and heat-shock proteins. The synthesis rates of heat-shock proteins and constitutive plasmid-encoded proteins increased after the temperature upshift also in the control strain without hFGF-2 gene but followed time profiles different from the producing strain. The energy demand for the extra synthesis of plasmid-encoded and heat-shock proteins resulted in an elevated respiratory activity and, consequently, in a reduction of the growth rate and the biomass yield. A procedure was developed to relate the energy demand for the additional synthesis of these proteins to the generation of energy in the respiratory pathway. Specific energy production was estimated based on on-line measurable rates of oxygen consumption, or carbondioxide evolution and growth, respectively. In this way, the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins was quantified from on-line accessible data.
Hoffmann
F
F
Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Rinas
U
U
D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006801:Humans; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption; D010957:Plasmids / Q000378:metabolism*; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D010100:Oxygen