Using high-cell-density culture of Escherichia coli under the control of an L-arabinose promoter (ParaB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied. The inducer, L-arabinose, showed a maximal induction level above 10.7 mM in the final concentration. The concentration of inducer also affected the partition of interferon-alpha (IFN-alpha) into the soluble form and inclusion bodies. Induction kinetics of the rate of accumulation of IFN-alpha on the ParaB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac. These innate characteristics of ParaB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein. The duration time of induction could control the expression of both soluble and insoluble protein. The ratio of yeast extract to glycerol (N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation. The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion bodies in the total expressed IFN-alpha.
To develop the optimal operational strategy for the PL promoter of interferon (IFN)-gamma-producing Escherichia coli, some rational medium feeding methods, carried out just after temperature induction to control the postinduction specific growth rate (mu i), were designed. Using experimental data from various batch cultures, we correlated mu i with specific IFN-gamma production rate (qpi) to find out how mu i just after induction affected qpi. It was revealed that sustaining mu i above a certain value after induction was the key factor for high-level production of IFN-gamma. Stepwise and constant-rate medium feeding, in which the temperature induction was done simultaneously, at the late-exponential growth phase of batch culture resulted in a dramatic extension of the production period as a result of sustaining mu i after temperature induction. These methods led to the overcoming of harsh conditions after temperature induction and the improvement of IFN-gamma productivity. Finally, by both a high-cell-density culture using an exponential fed-batch culture for cell growth and constant-rate medium feeding after temperature induction for IFN-gamma production, we accomplished an increase in IFN-gamma production by 23-fold, 7.43 g/L, as compared with that of batch culture.
A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h(-1) produced a maximum hirudin concentration of 62.1 mg l(-1), which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.
A DO-stat control strategy for two variables was introduced to the rGuamerin production process in Pichia pastoris and applied to repeated fed-batch culture. Two interrelated variables, namely the ratio of partial pressure of pure O2 in the inlet air-stream and the methanol feed rate, were controlled simultaneously. By using this control strategy, methanol feeding for induction could be controlled automatically while efficiently controlling the dissolved oxygen level. As a result, the cell concentration reached more than 140 g l(-1) and rGuamerin expression level 450 iu l(-1). rGuamerin was secreted into the culture medium and reached a level that was 40% higher than achieved in a fed-batch process using manual control of the methanol feeding rate. Repeated rGuamerin induction was achieved by repeating the methanol feeding and withdrawing the culture broth during extended production. During more than 250 h of culture, expression of rGuamerin was maintained at an average of about 430 iu l(-1 )(473 mg l(-1)), without causing the cell density to decrease. In addition to the rGuamerin production process, the proposed control system might be applied to cultivation of other methylotrophic yeasts in the production of therapeutic proteins.
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