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Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies

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Structural Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2305))

Abstract

Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.

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Acknowledgments

This work was funded by Instruct-ULTRA (Coordination and Support Action Number ID 731005) funded by the EU H2020 framework to further develop the services of Instruct-ERIC and the Wellcome Trust Grants 202892/Z/16/Z (Membrane Protein Laboratory) and 090532/Z/09/Z (Wellcome Human Genetics Centre).

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Authors and Affiliations

  1. Division of Structural Biology, University of Oxford, The Wellcome Centre for Human Genetics, Headington, Oxford, UK

    Ganna O. Krasnoselska & Raymond J. Owens

  2. The Rosalind Franklin Institute, Didcot, UK

    Maud Dumoux & Raymond J. Owens

  3. Diamond Light Source Ltd, Didcot, UK

    Nadisha Gamage, Harish Cheruvara, James Birch & Andrew Quigley

Authors
  1. Ganna O. Krasnoselska
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  2. Maud Dumoux
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  3. Nadisha Gamage
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  4. Harish Cheruvara
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  5. James Birch
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  6. Andrew Quigley
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  7. Raymond J. Owens
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Corresponding author

Correspondence to Raymond J. Owens .

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Editors and Affiliations

  1. The Rosalind Franklin Institute, Harwell Science Campus, Didcot, UK

    Raymond J. Owens

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Krasnoselska, G.O. et al. (2021). Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies. In: Owens, R.J. (eds) Structural Proteomics. Methods in Molecular Biology, vol 2305. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1406-8_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1406-8_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1405-1

  • Online ISBN: 978-1-0716-1406-8

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