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Npro fusion technology to produce proteins with authentic N termini in E. coli

Nature Methods volume 4, pages 1037–1043 (2007)Cite this article

Abstract

We describe a prokaryotic expression system using the autoproteolytic function of Npro from classical swine fever virus. Proteins or peptides expressed as Npro fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus. A tailor-made Npro mutant called EDDIE, with increased in vitro and decreased in vivo cleavage rates, has enabled us to express proinsulin, domain-D of staphylococcal protein A, hepcidin, interferon-α1, keratin-associated protein 10-4, green fluorescent protein, inhibitorial peptide of senescence-evasion-factor, monocyte chemoattractant protein-1 and toxic gyrase inhibitor, among others. This Npro expression system can be used as a generic tool for the high-level production of recombinant toxic peptides and proteins (up to 12 g/l) in Escherichia coli without the need for chemical or enzymatic removal of the fusion tag.

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Figure 1: Protein expression by the Npro system.
Figure 2: Expression of Npro fusion proteins and peptides.
Figure 3: EDDIE shows improved refolding properties relative to Npro.

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Acknowledgements

We acknowledge the contributions of Sandoz GmbH (P. Alliger, M. Frank, K. Graumann, F. Hochholdinger, S. Keller, B. Knorr, A. Krämer, A. Plematl, D. Serp, G. Stoller and W. Walcher). We thank M. Gudelj-Wyletal, D. Keller and M. Füreder (Boehringer Ingelheim Austria) for expression vector construction, fermentation and refolding development of 6His-EDDIE-rhMCP-1; the group of Herbert Lindner for N-terminal sequencing; D. Kolarich for mass spectrometric analysis; H. Zoller for cooperation concerning Hepcidin; and R. Schneider for discussion and ideas. This work was performed within the Austrian Center of Biopharmaceutical Technology (ACBT) a competence center funded by the Austrian Ministry of Economics and Labor, the federal states of Vienna and Tyrol and by its industrial partners Sandoz GmbH and Boehringer Ingelheim Austria GmbH.

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Authors and Affiliations

  1. Austrian Center of Biopharmaceutical Technology, Muthgasse 18, Vienna, A-1190, Austria

    Clemens Achmüller, Waltraud Kaar, Karin Ahrer, Philipp Wechner, Rainer Hahn, Florian Werther, Hannes Schmidinger, Monika Cserjan-Puschmann, Franz Clementschitsch, Gerald Striedner, Karl Bayer, Alois Jungbauer & Bernhard Auer

  2. Institute of Biochemistry, University of Innsbruck, Peter Mayr Strasse 1a, Innsbruck, A-6020, Austria

    Clemens Achmüller, Philipp Wechner, Florian Werther & Bernhard Auer

  3. Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, Vienna, A-1190, Austria

    Waltraud Kaar, Karin Ahrer, Rainer Hahn, Monika Cserjan-Puschmann, Franz Clementschitsch, Gerald Striedner, Karl Bayer & Alois Jungbauer

  4. Sandoz GmbH, Biochemiestrasse 10, A-6250, Kundl, Austria

    Philipp Wechner & Florian Werther

  5. Boehringer Ingelheim Austria GmbH, Dr.-Boehringer-Gasse 5-11, Vienna, A-1121, Austria

    Hannes Schmidinger

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  1. Clemens Achmüller
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Contributions

C.A., P.W. and F.W. optimized the Npro fusion concept, generated mutants and fusion proteins, and examined in vivo and in vitro cleavage. W.K., K.A., R.H. and H.S. analyzed in vitro refolding of fusion proteins, determined the kinetic constants of the cleavage reaction, and developed protein purification methods. G.S., M.C.-P., and F.C. performed fermentations of Npro fusions. C.A., K.A. and R.H. drafted the manuscript. B.A., A.J. and K.B. were responsible for the concept of expression and processing heterologous proteins in E. coli in fusion with Npro and revised the manuscript.

Corresponding author

Correspondence to Bernhard Auer.

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Competing interests

P. W. and F. W. are employees of Sandoz GmbH (Kundl, Austria) and H. S. of Boehringer Ingelheim Austria GmbH (Vienna). Npro-fusion technology is proprietary to Boehringer Ingelheim Austria GmbH and Sandoz GmbH, but will be available for research purposes upon signing a license agreement and sending it back to the corresponding author. A respective form is available online (Supplementary Note).

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Supplementary Figures 1–4, Supplementary Tables 1–2, Supplementary Methods, Supplementary Protocol (PDF 826 kb)

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Achmüller, C., Kaar, W., Ahrer, K. et al. Npro fusion technology to produce proteins with authentic N termini in E. coli. Nat Methods 4, 1037–1043 (2007). https://doi.org/10.1038/nmeth1116

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