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Bacterial Secretion Systems for Use in Biotechnology: Autotransporter-Based Cell Surface Display and Ultrahigh-Throughput Screening of Large Protein Libraries

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Hydrocarbon and Lipid Microbiology Protocols

Part of the book series: Springer Protocols Handbooks ((SPH))

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Abstract

In recent years, continuous progress was made in our understanding of bacterial secretion pathways and the application of protein secretion for biotechnology. Efficient protein export is a prerequisite for cost-effective downstream processing, and secretion of a protein of interest may also be useful for certain enzyme assays, for biotransformation reactions, and, in particular, for screening enzyme variants in libraries generated by directed evolution. Cells that display a particular enzyme variant can be exposed to a broad spectrum of different chemical environments, can sustain a broad pH range, and can therefore allow one to probe the desired target protein under defined conditions. In recent years, fluorescent-activated cell sorting (FACS) in combination with cell surface display has become a powerful tool for the activity-based ultrahigh-throughput screening of mutant proteins from large libraries. The below protocol provides the detailed description for the generation of protein libraries, E. coli cell surface display, and library screening using magnetic- and fluorescence-activated cell sorting technologies with an emphasis on activity and selectivity screening of lipases and esterases.

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Author information

Authors and Affiliations

  1. Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

    Karl-Erich Jaeger

  2. Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52426, Jülich, Germany

    Karl-Erich Jaeger

  3. Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, 64287, Darmstadt, Germany

    Harald Kolmar

Authors
  1. Karl-Erich Jaeger
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  2. Harald Kolmar
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Corresponding author

Correspondence to Harald Kolmar .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

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Jaeger, KE., Kolmar, H. (2015). Bacterial Secretion Systems for Use in Biotechnology: Autotransporter-Based Cell Surface Display and Ultrahigh-Throughput Screening of Large Protein Libraries. In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_125

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  • DOI: https://doi.org/10.1007/8623_2015_125

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-53113-6

  • Online ISBN: 978-3-662-53115-0

  • eBook Packages: Springer Protocols

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