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Optimization of Membrane Protein Production Using Titratable Strains of E. coli

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Heterologous Gene Expression in E.coli

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1586))

Abstract

The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.

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Acknowledgments

The Oxford Protein Production Facility-UK is supported by the UK Medical Research Council (MR/K018779/1) and the Manchester Group are supported by the Biotechnology and Biology Research Council, ND holds a BBSRC David Phillips Fellowship [BB/K014773/1].

Author information

Authors and Affiliations

  1. Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK

    Rosa Morra, Kate Young & Neil Dixon

  2. Research Complex at Harwell, Rutherford Appleton Laboratory, Oxfordshire, OX11 0FA, UK

    David Casas-Mao

  3. School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK

    David Casas-Mao

  4. Oxford Protein Production Facility-UK, Research Complex at Harwell, Rutherford Appleton Laboratory, Oxfordshire, OX11 0FA, UK

    Louise E. Bird

  5. Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK

    Louise E. Bird

Authors
  1. Rosa Morra
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  2. Kate Young
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  3. David Casas-Mao
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  4. Neil Dixon
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  5. Louise E. Bird
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Corresponding authors

Correspondence to Neil Dixon or Louise E. Bird .

Editor information

Editors and Affiliations

  1. SGC, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

    Nicola A. Burgess-Brown

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Morra, R., Young, K., Casas-Mao, D., Dixon, N., Bird, L.E. (2017). Optimization of Membrane Protein Production Using Titratable Strains of E. coli . In: Burgess-Brown, N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6887-9_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6887-9_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6885-5

  • Online ISBN: 978-1-4939-6887-9

  • eBook Packages: Springer Protocols

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