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Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants

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Abstract

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.

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Acknowledgments

This work was supported by an Ernst-von-Leyden Stipendium of the Berliner Krebsgesellschaft to Vânia Coelho and by Deutsche Krebshilfe grant no. 1072981 to PMD.

Competing interests

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Medizinische Klinik III, Hematology, Oncology und Transfusion Medicine, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin, Germany

    Hossein Panjideh, Vânia Coelho, Ulrich Keilholz, Eckhard Thiel & P. Markus Deckert

  2. Institute for Clinical Chemistry and Pathobiochemistry (JD, HF), Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, 12200, Berlin, Germany

    Jens Dernedde & Hendrik Fuchs

Authors
  1. Hossein Panjideh
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  2. Vânia Coelho
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  3. Jens Dernedde
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  4. Hendrik Fuchs
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  5. Ulrich Keilholz
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  6. Eckhard Thiel
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  7. P. Markus Deckert
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Correspondence to P. Markus Deckert.

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Panjideh, H., Coelho, V., Dernedde, J. et al. Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants. Bioprocess Biosyst Eng 31, 559–568 (2008). https://doi.org/10.1007/s00449-008-0203-y

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  • DOI: https://doi.org/10.1007/s00449-008-0203-y

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