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Improvement of Porcine Interferon-α-production Performance and ATP Efficiency at Reducing Inducion Temperature

Author: LiuGuoQiang
Tutor: ShiZhongPing
School: Jiangnan University
Course: Fermentation Engineering
Keywords: Pichia pastoris Hypothermia ATP Metabolic flux Methanol than the rate of consumption Porcine interferon-α
CLC: TQ929.1
Type: Master's thesis
Year: 2010
Downloads: 27
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Abstract

In this thesis recombinant Pichia pastoris strain (Pichia psatoris) KM71H (IFNα-pPICZαA) mode strain expression the pig alpha interferon (pIFN-α) shake the bottle and 10L fermenter conditions, effects of environmental factors on pIFN- α concentrations and antiviral activity. The flask experiment results show that the temperature on the fermentation performance of pIFN-α significantly affected. 10L fermenter, combined with the the Pichia pastoris use methanol simplified metabolic model state parameters by monitoring the fermentation process, a comprehensive analysis of the effect of temperature on the performance of pIFN-α production. The main contents of the paper and the results are as follows: (1) Select the induction temperature, induction of methanol concentration and pH of the investigated factors, experimental design, the use of shake-flask cultures of Pichia yeast production the expression pIFN-α. Induced 72h, the supernatant by SDS-PAGE analysis of the results showed that the 20 ° C low temperature induced pIFN-α protein bands was significantly thicker than 30 ° C induced bands, there is conducive to the expression of pIFN-α production induced by low temperature. Using gel image analysis of the scanned image, the induction temperature of 20 ° C, the methanol concentration 20g / L, pH = 5.5 under the conditions of the highest concentration of pIFN-α up to 0.19g / L. (2) In the 10L fermenter and different induction temperatures, high density culture of Pichia pastoris production pIFN-α fermentation experiment. Use of online monitoring instruments commonly used in industrial production, on-line monitoring of the state of fermentation process parameters (methanol consumption rate, oxygen consumption rate OUR CO2 generation rate of CER, respiratory quotient RQ) found that the above parameters are quite different in the different induction temperatures . 20 ℃, the high activity of cell metabolism, the methanol is faster than the rate of consumption. Combined with simplified metabolic model cells use methanol metabolic analysis, the results showed that the change the induced temperature can lead to intracellular production capacity pathway (pathway A) and the target substance synthesis pathway (pathway B) changes in metabolic flux distribution ratio. Induced by low temperature 20 ° C under the reinforced carbon metabolism liquidity biosynthetic pathway towards pIFN-α, compared with 30 ℃ induced fermentation 90 h when the metabolic flux ratio of 67%. Pichia pastoris methanol (3) the use of generally accepted substrate simplifies the metabolic model, combined with online measurement of the state parameters of the fermentation process, as well as of intracellular oxidative phosphorylation pathway enzymes (NADH / NAD) and the high-energy phosphate compounds (ATP , ADP, AMP) detection, pIFN-α expression energy production system operation mechanism. Analysis calculated that the 20 ° C low-temperature induction conditions, capacity pathway of NADH generated can be effectively utilized in the product synthesis pathway, the biosynthesis pathway required ATP energy regeneration efficiency increase ATP reproduction speed is increased by 6 - 24%, the fermentation of Pichia the overall efficiency of the production of exogenous proteins to be improved. (4) 10L fermenter highest pIFN-α activity in the 20 ° C inducing conditions up to 1.5 × 106IU/mL, the same volume fermenter 30 ° C induced production of 150 times the highest activity (1 × 104IU/mL).

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