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Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris

  • Applied Genetics and Molecular Biotechnology
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Abstract

In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l−1 in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.

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Acknowledgements

We would like to thank Dr. Charles K. Barlowe from Dartmouth University for providing the anti-Kar2p for our BiP analyses. This project was funded by the Ludwig Institute for Cancer Research.

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Authors and Affiliations

  1. Department of Microbiology, Cornell University, Ithaca, NY, 14853, USA

    Leonardo M. Damasceno & Kyle A. Anderson

  2. Department of Food Science, Cornell University, 317 Stocking Hall, Ithaca, NY, 14853, USA

    Carl A. Batt

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

    Gerd Ritter & Lloyd J. Old

  4. Keck Graduate Institute, 535 Watson DriveClaremont, Claremont, CA, 91711, USA

    James M. Cregg

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  1. Leonardo M. Damasceno
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  2. Kyle A. Anderson
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  3. Gerd Ritter
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  4. James M. Cregg
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  5. Lloyd J. Old
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  6. Carl A. Batt
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Correspondence to Leonardo M. Damasceno or Carl A. Batt.

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Damasceno, L.M., Anderson, K.A., Ritter, G. et al. Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris . Appl Microbiol Biotechnol 74, 381–389 (2007). https://doi.org/10.1007/s00253-006-0652-7

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