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Production of recombinant protein in Pichia pastoris by fermentation

Nature Protocols volume 1, pages 1006–1021 (2006)Cite this article

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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Figure 1: Process flow scheme.
Figure 2: Agitation and DO traces during glycerol to methanol conversion.
Figure 3: Example of a pump head of a peristaltic limiting feed pump.
Figure 4: Biomass (black), MeOH flow rate (red) and product formation (blue), during fermentation with 72 h EFT.

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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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Authors and Affiliations

  1. Department of Oncology, Royal Free and University College Medical School, University College London, Rowland Hill Street, London, NW3 2PF, UK

    Berend Tolner, Lisa Smith, Richard H J Begent & Kerry A Chester

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  1. Berend Tolner
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  2. Lisa Smith
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  3. Richard H J Begent
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  4. Kerry A Chester
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Corresponding author

Correspondence to Berend Tolner.

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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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