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A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation

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Abstract

We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins.

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Figure 1: Schematic representation of the protein expression and glycosylation engineering system.
Figure 2: Production of homogenous N-glycoproteins.

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Change history

  • 18 March 2010

    In the version of this article initially published online, an extra blue square corresponding to a GlcNAc monomer was shown in the transglycosylation step in Figure 1 and the Graphical Abstract. Also, panels g and h of Figure 2 were mistakenly swapped. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank members of the Aebi and Wang labs for fruitful discussions. This work was supported in part by the Swiss National Science Foundation (grant 3100AQ-105541 to M.A.), the Eidgenössische Technische Hochschule Zurich and the US National Institutes of Health (grant GM080374 to L.-X.W.). We thank K. Takegawa (Kyushu University) for providing the pGEX-2T/Endo-A plasmid and K. Yamamoto (Kyoto University) for providing the pET23b-Endo-M plasmid, which were used to express the endo enzymes. F.S. and C. Lizak are members of the Zurich PhD Program in Molecular Life Sciences.

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Authors and Affiliations

  1. Department of Biology, Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland

    Flavio Schwarz, Benjamin L Schulz, Christian Lizak, Shin Numao & Markus Aebi

  2. Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA

    Wei Huang, Cishan Li & Lai-Xi Wang

  3. Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA

    Wei Huang, Cishan Li & Lai-Xi Wang

  4. Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland

    Alessandro Palumbo & Dario Neri

Authors
  1. Flavio Schwarz
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  2. Wei Huang
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  3. Cishan Li
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  4. Benjamin L Schulz
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  5. Christian Lizak
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  6. Alessandro Palumbo
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  7. Shin Numao
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  8. Dario Neri
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  9. Markus Aebi
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  10. Lai-Xi Wang
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Contributions

F.S. engineered the glycosylation pathway, characterized proteins and wrote the manuscript. W.H. trimmed and remodeled glycans and characterized products. C. Li remodeled glycans and characterized products. B.L.S. performed preliminary MS analyses of glycosylated AcrA. C. Lizak engineered the CH2 domain. A.P. characterized the F8 protein. S.N. conceived the original idea and performed preliminary experiments. D.N. developed F8 and supervised the research. M.A. supervised the research and wrote the manuscript. L.-X.W. conceived the original idea, supervised the research and wrote the manuscript. All authors contributed to editing the manuscript.

Corresponding authors

Correspondence to Markus Aebi or Lai-Xi Wang.

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Competing interests

D.N. is a co-founder and shareholder of Philogen, the company that owns the F8 antibody.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Results, and Supplementary Figures 1–9 (PDF 645 kb)

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Schwarz, F., Huang, W., Li, C. et al. A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat Chem Biol 6, 264–266 (2010). https://doi.org/10.1038/nchembio.314

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  • DOI: https://doi.org/10.1038/nchembio.314

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