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Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct

Authors

  • Christian Berger,

    1. Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, Leipzig 04107, Germany; telephone: 0341-9715701; fax: 0341-9715709
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  • Sandra Berndt,

    1. Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, Leipzig 04107, Germany; telephone: 0341-9715701; fax: 0341-9715709
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  • Annelie Pichert,

    1. Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, Leipzig 04107, Germany; telephone: 0341-9715701; fax: 0341-9715709
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  • Stephan Theisgen,

    1. Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, Leipzig 04107, Germany; telephone: 0341-9715701; fax: 0341-9715709
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  • Daniel Huster

    Corresponding author
    1. Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, Leipzig 04107, Germany; telephone: 0341-9715701; fax: 0341-9715709
    • Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, Leipzig 04107, Germany; telephone: 0341-9715701; fax: 0341-9715709.
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Abstract

A protocol for the efficient isotopic labeling of large G protein-coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L-tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell–cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell–cell communication by the addition of indole during expression. Discrete concentrations of indole and 15N2-L-tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ∼15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681–1690. © 2013 Wiley Periodicals, Inc.

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