Studies On Fermentation Of Human Epidermal Growth Factor In Recombinant Escherichia Coli Considering Cell Physiology

The expression of recombinant protein in Escherichia coli consumes intracellular resources and exerts extra metabolic burden on the host cell. As a result, the imposition of over-weighted metabolic burden would alter and even impair the functioning and metabolism of host cell. At the same time, stress responses occur in E. coli during the expression of recombinant protein to rebalance the cell metabolism. For the sake of over-expression of target protein, cell physiological and metabolism should be considered in the optimization process.The purpose of this work is to investigate the effect of recombinant human epidermal growth factor (hEGF) expression on cell physiology in E. coli, and provide a scientific basis for optimization of recombinant protein expression.The growth inhibition of the recombinant E. coli in the expression of hEGF was observed. It was found that the recombinant cells segregated into plasmid-free cells and viable but non-culturable cells in the post-induction phase. Chromosome DNA relaxation was observed with transmitted electron microscopy during the recombinant protein expression. The DNA relaxation was proposed to correlate with impairment of chromosome DNA and could result in the decrease of dividing ability in E. coli. A colorimetric assay was carried out to measure the dehydrogenase activity of respiratory chain in E. coli, and it was found that the cell respiratory ability was inhibited in the post-induction phase of recombinant protein expression. Furthermore, the reason of extracellular proteins production and the effect of hEGF expression on extracelluar proteins production were also studied.Based on the cell ability for cell division and plasmid maintenance, the recombinant cells could be segregated into three populations: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable but non-culturable cells. The fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and recombinant protein production. The results showed that low concentration of inducer caused weak induction, whereas high levels would cause strong induction resulting in cells segregation into viable but non-culturable bacteria and low foreign protein yield. A kinetic model considering cell segregation was proposed and fitted well with the experimental data of cell growth andprotein expression. In addition, the optimal induction strategy could be predicted by the model, and was verified by the experiments with maximum expression of recombinant protein.Relations between dissolved oxygen oscillation and pulses in the glucose feed rate in fed-batch cultivation of recombinant E. coli were characterized. The dissolved oxygen response signal was used to detect the acetic acid production in pulsed fed-batch cultivation of E. coli. In fed-batch cultivation with glucose-limited growth at specific growth rate 0.1 h"1, acetic acid was kept in low level and dissolved oxygen response signal was oscillated along with the pulses in the glucose feed rate in fed-batch phase. The critical specific oxygen uptake rate and the critical specific glucose uptake rate were reduced after induction of recombinant human epidermal growth factor. Consequently, acetic acid was readily accumulated in the medium and the dissolved oxygen response signal lost its oscillation characteristics. Using feedback control of dissolved oxygen signal, the glucose feed rate in post-induction phase was effectively manipulated and acetic acid accumulation was avoided successfully. Cell concentration of 48 g L"1 dry cell weight was obtained. The level of 0.181 g L"1 human epidermal growth factor was gained and inproved 45 % comparing that in fed-batch cultivation without feedback control.The effect of tryptone addition on cell physiology in pulsed fed-batch cultivation of E. coli was investigated. Applying the feedback control of dissolved oxygen signal responding pulsed in the glucose feed rate, the acetic acid production was successfully prevented and the optimization for recombinant hEGF pro...