Study On The Metabolic And Physiological Characteristics Of Recombinant Pichia Pastoris Producing A Fusion Protein

The characteristics of recombinant Pichia pastoris fermentation process producing HSA-IL2 in a 5 L fermentor were explored. Based on the on-line and off-line measurements, as well as the method of correlation analysis, the activities of three key enzymes (i.e. alcohol oxidase, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase) were characterized with oxygen uptake rate and carbon dioxide evolution rate. The results showed that: (1) the activities of glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase were in direct proportion to the cell specific growth rate(μ) in cell growth phase; (2) The activities of glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase sharply decreased in the methanol induction phase, indicating that metabolic flux shift occurred during fermentation process; (3) The changes of alcohol oxidase activity and HSA-IL2 expression exhibited different patterns with different control strategies. With the control strategy of higher dissolved oxygen (DO) and lower methanol concentration, the final HSA-IL2 expression amount reached 27 mg/L, which was 33.7% higher than that achieved with the control strategy of lower DO and higher methanol concentration.The method of determining AMP, ADP, ATP, NAD+ and NADH in recombinant Pichia pastoris producing a fusion protein by high performance liquid chromatography(HPLC) was established and the analysis process was less than 15 min. By HPLC analysis the profiles of both of energy charge and oxidationte-reduction state in vivo throughout the fermentation were studied.The results showed that: (1) the concentration of AMP, ADP, ATP, NAD+ and NADH in vivo was regulated by recombinant Pichia pastoris itself through energy charge and oxidationte-reduction state and it led to corresponding metabolic flux changes which had an effect on the cell growth and the biosynthesis of fusion protein; (2) PCA analysis showed that the respiration characteristics of recombinant Pichia pastoris reflected the cell physiological state to some extent.