Semi-continuous microcarrier culture of rCHO cells secreting HBsAg by feeding microcarriers.

On the basis of culturing rCHO cells semi-continuously with a constant concentration of microcarriers, cell yield and HBsAg expression were increased by feeding microcarriers, step by step, to provide a surface for cells to grow. A process of culturing rCHO cells semi-continuously to produce HBsAg by feeding microcarriers was established, in which the maximum microcarrier concentration was defined. The foundation has been laid for culturing rCHO cells continuously to reach high cell yield and high HBsAg expression.

Study on growth kinetics of unstable recombinant Saccharomyces cerevisiae.

A mathematical model is suggested to describe effects of intracellular inhibition on growth of the recombinant yeast cells. The inhibition of the growth of the Leu+ phenotype cells depends upon the increase of plasmid concentration, the copy numbers and the accumulated amount of hepatitis B surface antigen protein, the expressed gene product. Simulating the growth of plasmid-free cells gives possible age-time distribution corresponding to their formation. According to the growth trajectories of these two different phenotype cells, a relation between Leu- phenotype cell growth and recombinant plasmid loss frequency is obtained. Consequently the instability of recombinant plasmid appearing in fermentation by genetically engineered S. cerevisiae is affected mainly due to (1) regular partitioning of Leu- phenotype combined with asymmetric plasmid segregation, and (2) different growth rates of plasmid-containing and plasmid-free cells in complex medium.

Effect of pH value and dissolved oxygen concentration on expression of hepatitis B surface antigen by genetically engineered yeast in fermentation processes.

Both cell growth and expression of hepatitis B surface antigen (HBsAg) interact with each other in fermentation to produce exogenous protein HBsAg by genetically engineered yeast, S. cerevisiae, which contains the HBsAg gene. Since both processes required proper pH values in order to improve the overall yield, a suitable pH value sequence was determined. It is important that dissolved oxygen concentration in fermentation should be controlled to maintain the stability of recombinant plasmids and to obtain the high HBsAg expression. When the concentration of dissolved oxygen was controlled at 70% saturation, the stability of recombinant plasmid was maintained at 73%, and the relative concentration of HBsAg expression was found to be 98.6.