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A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures

  • Genomics, Transcriptomics, Proteomics
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The secreted proteome of Pichia pastoris X-33 was investigated in methanol-induced cultures with a goal to enhance the secretion and purification of recombinant proteins. In a fed-batch fermentation at 30 °C, more host proteins were found in greater concentrations compared to cultures grown at 25 °C. Protein samples collected directly from the culture media at 25 °C, as well as separated by two-dimensional (2D) gel, were subjected to ESI-MS/MS analysis. A total of 75 proteins were identified in the media from different conditions including pre- and post-methanol induction and in a strain overexpressing a recombinant schistosomiasis vaccine, Sm14-C62V. The identified proteins include native secreted proteins and some intracellular proteins, most of which have low isoelectric points (pI < 6). 2D gel analyses further revealed important characteristics, such as abundance, degradation, and glycosylation of these identified proteins in this proteome. Cell wall-associated proteins involved in cell wall biogenesis, structure, and modification comprised the majority of the secreted proteins which have been identified. Intracellular proteins such as alcohol oxidase and superoxide dismutase were also found in the proteome, suggesting some degree of cell lysis. However, both protocols show that their concentrations are significantly lower than the native secreted proteins. This study identifies proteins secreted or released into the culture media in the methanol-induced fermentation cultures of P. pastoris X-33 and suggests potential biotechnology applications based on the discovery of this proteome.

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Acknowledgements

The authors thank Mrs. Sabine Baumgart for mass sample preparation and invaluable suggestions for the study. This work was supported by Cornell University/Ludwig Institute for Cancer Research Partnership.

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Authors and Affiliations

  1. Field of Microbiology (317 Stocking Hall), Cornell University, Ithaca, NY, 14853, USA

    Chung-Jr Huang, Leonardo M. Damasceno, Kyle A. Anderson & Carl A. Batt

  2. Proteomic and Mass Spectrometry Facility, Cornell University, Ithaca, NY, 14853, USA

    Sheng Zhang

  3. Ludwig Institute for Cancer Research (New York Branch), Memorial Sloan-Kettering Cancer Center, New York, NY, 10021, USA

    Lloyd J. Old

  4. School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA

    Chung-Jr Huang

  5. Sanofi Pasteur, Swiftwater, PA, 18370, USA

    Leonardo M. Damasceno

  6. Tetragenetics, Ithaca, NY, 14859, USA

    Kyle A. Anderson

Authors
  1. Chung-Jr Huang
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  2. Leonardo M. Damasceno
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  3. Kyle A. Anderson
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  4. Sheng Zhang
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  5. Lloyd J. Old
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  6. Carl A. Batt
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Corresponding author

Correspondence to Carl A. Batt.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplement 1

(XLS 356 kb)

Supplement 2

Table A. The Blast results of identified P. pastoris proteins against S. cerevisiae protein database (DOC 123 kb)

Supplement 3

(XLS 1 kb)

Supplement 4

Identification of proteins in 23 2D gel spots showing high protein scores and at least two peptide hits (DOC 119 kb)

Supplement 5

Glycostain of the Sm-14 gel. A glycostain (A) and a Coomassie stain (B) of the Sm14 gel for identification of glycoprotein in the gel spots. The spot numbers are shown based on Fig. 4. The square indicates leftover dye on the 2D gel. (JPEG 46 kb)

Supplement 6

Comparison of identified proteins in glucose-induced culture (Mattanovich et al. 2009b) to their protein homologs identified from this study (DOC 44 kb)

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Huang, CJ., Damasceno, L.M., Anderson, K.A. et al. A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. Appl Microbiol Biotechnol 90, 235–247 (2011). https://doi.org/10.1007/s00253-011-3118-5

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  • DOI: https://doi.org/10.1007/s00253-011-3118-5

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