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Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained at 25–30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins.

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Authors and Affiliations

  1. Institute of Tropical Bioscience and Biotechnology/National Key Biotechnology Laboratory for Tropical Crops, CATAS, Haikou Hainan, 571101, China

    A. L. Zhang, S. C. Chen, S. Q. Peng & H. L. Li

  2. The Key Laboratory of Gene Engineering of Ministry of Education and Department of Biochemistry, Sun Yat-Sen University, Guangzhou Guangdong, 510275, China

    T. Y. Zhang, J. X. Luo & W. J. Guan

  3. Hainan Provincial Institute for Drug Control, Haikou Hainan, 570216, China

    C. Y. Fu

Authors
  1. A. L. Zhang
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  2. T. Y. Zhang
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  3. J. X. Luo
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  4. S. C. Chen
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  5. W. J. Guan
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  8. H. L. Li
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Correspondence to A. L. Zhang.

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Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal Government of China (99-Z-004-001).

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Zhang, A.L., Zhang, T.Y., Luo, J.X. et al. Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture. J Ind Microbiol Biotechnol 34, 117–122 (2007). https://doi.org/10.1007/s10295-006-0175-3

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  • DOI: https://doi.org/10.1007/s10295-006-0175-3

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