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中国生物工程杂志

China Biotechnology
China Biotechnology  2015, Vol. 35 Issue (10): 39-43    DOI: 10.13523/j.cb.20151006
    
The Effects of Carbon Source on High-density Fermentation of Pichia pastoris and HPV16_L1 Expression under the Control of Constitutive TEF-1 Promoter
XIA Ye1,2,3, HUANG Wei-wei1,2,3, YANG Xu1,2,3, SUN Peng-yan1,2,3, YAO Yue-ting1,2,3, WANG Shi-jie1,2,3, LIU Cun-bao1,2,3, SUN Wen-jia1,2,3, BAI Hong-mei1,2,3, YAO Yu-feng1,2,3, MA Yan-bing1,2,3
1. Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China ;
2. Yunnan Key Laboratory of Research and Development on Severe Infection Disease, Kunming 650118, China;
3. Yunnan Engineering Research Center of Vaccine Research and Development on Severe Infectious Disease, Kunming 650118, China
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Abstract  

Objective:To investigate the effects of different carbon sources on the high density fermentation of Pichia pastoris and the expression of HPV16_L1 protein under the control of TEF-1 promoter. Methods:Pichia pastoris cells were cultured in 1.5L fermentors using media supplied respectively with a given carbon source. The dynamic curve of cell growth was obtained by sample collection and wet weight detection at designated time points, and Western blot analysis was employed to detect the expression level of HPV16_L1 protein. Results:Utilizing glycerol as a carbon source, the cells grew to a very high density with a cell wet weight of 510 g/L (OD600 was 189.4), and the expression of HPV L1 kept at a high level within 72 hours of fermentation. Utilizing methanol and dextran as carbon source, the wet weight of fermantation cells reached both to about 220g/L (OD600 values were 75.3 and 77.3 respectively). And in methanol utilization, the expression of L1 protein maintained at a high level during the 144 hours of fermentation; in contrast, in dextran utilization, L1 expression reduced significantly after 60 hours fermentation. Using sorbitol as solo carbon, the cells were not clearly proliferated in bioreactor. The highest yield was obtained by using glycerol as carbon source and the culture was collected after about 72 hours fermentation. Conclusion:The results provided valuable information for achieving high density fermentation of Pichia pastoris and efficient preparation of heterogeneous proteins expressed under the control of TEF-1 promoter, and also laid a foundation for developing HPV vaccine in a more convenient and economic way.



Key wordsPichia pastoris      High-density fermentation      TEF-1 promoter      Human papilloma virus major protein L1      Carbon source     
Received: 15 June 2015      Published: 25 October 2015
ZTFLH:  Q819  
Cite this article:

XIA Ye, HUANG Wei-wei, YANG Xu, SUN Peng-yan, YAO Yue-ting, WANG Shi-jie, LIU Cun-bao, SUN Wen-jia, BAI Hong-mei, YAO Yu-feng, MA Yan-bing. The Effects of Carbon Source on High-density Fermentation of Pichia pastoris and HPV16_L1 Expression under the Control of Constitutive TEF-1 Promoter. China Biotechnology, 2015, 35(10): 39-43.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20151006     OR     https://manu60.magtech.com.cn/biotech/Y2015/V35/I10/39

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