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Protein Engineering pp 145–156Cite as

Library Growth and Protein Expression: Optimal and Reproducible Microtiter Plate Expression of Recombinant Enzymes in E. coli Using MTP Shakers

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1685))

Abstract

Escherichia coli (E. coli) as heterologous host enables the recombinant expression of the desired protein in high amounts. Nevertheless, the expression in such a host, especially by utilizing a strong induction system, can result in insoluble and/or inactive protein fractions (inclusion bodies). Furthermore, the expression of different enzyme variants often leads to a diverse growth behavior of the E. coli clones resulting in the identification of false-positives when screening a mutant library. Thus, we developed a protocol for an optimal and reproducible protein expression in microtiter plates showcased for the expression of the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. By emerging this protocol, several parameters concerning the expression medium, the cultivation temperatures, shaking conditions as well as time and induction periods for CHMO were investigated. We employed a microtiter plate shaker with humidity and temperature control (Cytomat™) (integrated in a robotic platform) to obtain an even growth and expression over the plates. Our optimized protocol provides a comprehensive overview of the key factors influencing a reproducible protein expression and this should serve as basis for the adaptation to other enzyme classes.

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Authors and Affiliations

  1. Institute of Molecular Biotechnology, TU Graz, Graz, Austria

    Sandy Schmidt

  2. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, D-17487, Greifswald, Germany

    Mark Dörr & Uwe T. Bornscheuer

Authors
  1. Sandy Schmidt
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  2. Mark Dörr
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  3. Uwe T. Bornscheuer
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Corresponding author

Correspondence to Uwe T. Bornscheuer .

Editor information

Editors and Affiliations

  1. Department of Biotechnology and Enzyme Catalysis Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Uwe T. Bornscheuer

  2. Protein Biochemistry Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Matthias Höhne

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Schmidt, S., Dörr, M., Bornscheuer, U.T. (2018). Library Growth and Protein Expression: Optimal and Reproducible Microtiter Plate Expression of Recombinant Enzymes in E. coli Using MTP Shakers. In: Bornscheuer, U., Höhne, M. (eds) Protein Engineering. Methods in Molecular Biology, vol 1685. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7366-8_8

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  • DOI: https://doi.org/10.1007/978-1-4939-7366-8_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7364-4

  • Online ISBN: 978-1-4939-7366-8

  • eBook Packages: Springer Protocols

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