A secretory system for bacterial production of high-profile protein targets

Authors

  • Alexander Kotzsch,

    Corresponding author
    1. Facility for Protein Science and Technology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark
    • Alexander Kotzsch, University of Copenhagen, Faculty of Health Science, NNF Center for Protein Research, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

      Susanne Gräslund, Karolinska Institutet, Department of Medical Biochemistry and Biophysics, SGC/MBB, S-17177 Stockholm, Sweden

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  • Erik Vernet,

    1. Facility for Protein Science and Technology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark
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  • Martin Hammarström,

    1. Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Schéeles väg 2, 171 77 Stockholm, Sweden
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  • Jens Berthelsen,

    1. Facility for Protein Science and Technology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark
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  • Johan Weigelt,

    1. Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Schéeles väg 2, 171 77 Stockholm, Sweden
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  • Susanne Gräslund,

    Corresponding author
    1. Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Schéeles väg 2, 171 77 Stockholm, Sweden
    • Alexander Kotzsch, University of Copenhagen, Faculty of Health Science, NNF Center for Protein Research, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark

      Susanne Gräslund, Karolinska Institutet, Department of Medical Biochemistry and Biophysics, SGC/MBB, S-17177 Stockholm, Sweden

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  • Michael Sundström

    1. Facility for Protein Science and Technology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark
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Abstract

Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.

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