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High-Throughput Protein Production and Purification pp 191–202Cite as

High-Throughput Purification of Protein Kinases from Escherichia coli and Insect Cells

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

Abstract

Protein kinases are major targets for the development of new medicines and play key roles in cellular signaling. The flexible nature of these proteins, posttranslational modifications, and the large size of some protein kinases pose a particular challenge obtaining homogeneous, active recombinant protein kinases suitable for functional or structural studies. Here we describe our expertise expressing protein kinases in two frequently used host systems: E. coli and insect cells using the baculovirus expression vector system. In particular, we will discuss and provide detailed methods on construct design, high-throughput cloning, parallel expression testing and scale up as well as purification and co-expression strategies leading to stable and homogeneous recombinant protein samples.

Key words

  • Kinase expression
  • E. coli
  • Insect cells
  • Co-expression
  • Phosphatases

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Acknowledgements

The authors are grateful for financial support by the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, the Centre of Excellence (CEF) Macromolecular Complexes at Frankfurt University, and the Wellcome Trust. SK and SM are grateful for support by the German Cancer Centre (DKFZ) and the German Cancer Network (DKTK).

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Authors and Affiliations

  1. Target Discovery Institute and Structural Genomics Consortium, Oxford University, Oxford, UK

    Sebastian Mathea, Eidarus Salah & Stefan Knapp

  2. Goethe-University Frankfurt, Institute of Pharmaceutical Chemistry and Buchmann Institute for Life Sciences, Frankfurt am Main, Germany

    Sebastian Mathea & Stefan Knapp

  3. German Cancer Network (DKTK), Frankfurt am Main, Germany

    Sebastian Mathea & Stefan Knapp

  4. German Cancer Centre (DKFZ), Heidelberg, Germany

    Sebastian Mathea

Authors
  1. Sebastian Mathea
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  2. Eidarus Salah
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  3. Stefan Knapp
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Corresponding author

Correspondence to Stefan Knapp .

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Editors and Affiliations

  1. Architecture et Fonction des Macromolécules Biologiques (AFMB), Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Marseille cedex 9, France

    Dr. Renaud Vincentelli

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Mathea, S., Salah, E., Knapp, S. (2019). High-Throughput Purification of Protein Kinases from Escherichia coli and Insect Cells. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_8

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  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

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