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Expression and Purification of a Functional E. coli13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies

  • Fabian Bumbak
  • Ross A. D. Bathgate
  • Daniel J. Scott
  • Paul R. GooleyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)

Abstract

Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete 13CH3-methionine labeling of a thermostable neurotensin receptor 1 (enNTS1) variant in E. coli for solution NMR-based dynamics studies. Our expression strategy utilizes methionine biosynthesis pathway inhibition forcing E. coli to incorporate exogenous methionine with 96% efficiency at expression levels of 2.6 mg enNTS1 per liter of expression culture containing 50 mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6 mg of functional Apo-state enNTS1.

Key words

Membrane protein GPCR Neurotensin receptor 1 Thermostable E. coli 13CH3-methionine Apo-state 

Notes

Acknowledgments

This work was supported by NHMRC project grants 1081801 (D.J.S.) and 1081844 (R.A.D.B., P.R.G., D.J.S.); ARC equipment grant LE120100022. D.J.S. is an NHMRC Boosting Dementia Research Leadership Fellow. R.A.D.B. is an NHMRC Senior Research Fellow. Studies at The Florey Institute of Neuroscience and Mental Health were supported by the Victorian Government’s Operational Infrastructure Support Program.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Fabian Bumbak
    • 1
    • 2
    • 3
  • Ross A. D. Bathgate
    • 1
    • 3
  • Daniel J. Scott
    • 1
    • 3
  • Paul R. Gooley
    • 1
    • 2
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyThe University of MelbourneParkvilleAustralia
  2. 2.Bio21 Molecular Science and Biotechnology InstituteThe University of MelbourneParkvilleAustralia
  3. 3.The Florey Institute of Neuroscience and Mental HealthThe University of MelbourneParkvilleAustralia

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