Abstract
This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes ∼2 wk for each individual clone.
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Acknowledgements
The authors would like to thank J. Bhatia, N. Woods and T. Hillyer for contributions to this manuscript. This work is supported by Cancer Research UK, The Royal Free Cancer Research Trust, The Copley May Foundation and The National Translational Cancer Research Network (NTRAC).
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Supplementary information
Supplementary Note 1
Fermentor process log sheet (PDF 294 kb)
Supplementary Note 2
Aseptic handlings, Pichia spillage and disposal of waste (PDF 101 kb)
Supplementary Note 3
Calibration and maintenance of pH and DO probes (PDF 154 kb)
Supplementary Note 4
Guidelines for Pichia pastoris fermentation process development (PDF 127 kb)
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Tolner, B., Smith, L., Begent, R. et al. Production of recombinant protein in Pichia pastoris by fermentation. Nat Protoc 1, 1006–1021 (2006). https://doi.org/10.1038/nprot.2006.126
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DOI: https://doi.org/10.1038/nprot.2006.126
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