Chapter title |
Demonstration-Scale High-Cell-Density Fermentation of Pichia pastoris
|
---|---|
Chapter number | 9 |
Book title |
Recombinant Glycoprotein Production
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7312-5_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7311-8, 978-1-4939-7312-5
|
Authors |
Wan-Cang Liu, Ping Zhu |
Abstract |
Pichia pastoris has been one of the most successful heterologous overexpression systems in generating proteins for large-scale production through high-cell-density fermentation. However, optimizing conditions of the large-scale high-cell-density fermentation for biochemistry and industrialization is usually a laborious and time-consuming process. Furthermore, it is often difficult to produce authentic proteins in large quantities, which is a major obstacle for functional and structural features analysis and industrial application. For these reasons, we have developed a protocol for efficient demonstration-scale high-cell-density fermentation of P. pastoris, which employs a new methanol-feeding strategy-biomass-stat strategy and a strategy of increased air pressure instead of pure oxygen supplement. The protocol included three typical stages of glycerol batch fermentation (initial culture phase), glycerol fed-batch fermentation (biomass accumulation phase), and methanol fed-batch fermentation (induction phase), which allows direct online-monitoring of fermentation conditions, including broth pH, temperature, DO, anti-foam generation, and feeding of glycerol and methanol. Using this protocol, production of the recombinant β-xylosidase of Lentinula edodes origin in 1000-L scale fermentation can be up to ~900 mg/L or 9.4 mg/g cells (dry cell weight, intracellular expression), with the specific production rate and average specific production of 0.1 mg/g/h and 0.081 mg/g/h, respectively. The methodology described in this protocol can be easily transferred to other systems, and eligible to scale up for a large number of proteins used in either the scientific studies or commercial purposes. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 28 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Bachelor | 6 | 21% |
Student > Master | 5 | 18% |
Researcher | 2 | 7% |
Student > Ph. D. Student | 2 | 7% |
Lecturer | 1 | 4% |
Other | 2 | 7% |
Unknown | 10 | 36% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 10 | 36% |
Chemical Engineering | 4 | 14% |
Agricultural and Biological Sciences | 2 | 7% |
Chemistry | 1 | 4% |
Engineering | 1 | 4% |
Other | 0 | 0% |
Unknown | 10 | 36% |