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Noncanonical Amino Acids pp 169–190Cite as

Expression and Purification of Site-Specifically Lysine-Acetylated and Natively-Folded Proteins for Biophysical Investigations

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1728))

Abstract

N-(ε)-lysine-acetylation (short: lysine-acetylation) is a dynamic and powerful posttranslational modification to regulate protein function. Mutational approaches are often poor to access the real mechanistic impact of lysine-acetylation at the molecular level. Therefore, the ability to site-specifically incorporate N-(ε)-acetyl-l-lysine (short: AcK) into proteins dramatically increased our understanding how lysine-acetylation regulates protein function by using diverse molecular mechanisms going far beyond neutralizing a positive charge at the lysine-side chain. Genetically encoding AcK is a powerful way to introduce AcK into proteins, resulting in homogenously, quantitatively, and site-specifically lysine-acetylated proteins. Thereby, lysine-acetylated proteins can be produced in their natively-folded state in a high quality and in a yield sufficient to perform biophysical studies, including X-ray crystallography. This protocol describes the expression and purification of site-specifically lysine-acetylated proteins in Escherichia coli using the genetic-code expansion concept (GCEC) and subsequent steps to assess the successful incorporation of AcK by immunoblotting and mass-spectrometry.

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Acknowledgments

I thank Christian Frese from the CECAD proteomics facility for support in writing the mass-spectrometry section and Marcus Krüger critical reading of the mass-spectrometry part of this manuscript. Furthermore, I thank CECAD for support and all members of my laboratory, particularly Dr. Susanne de Boor, for the preparation of some figures and discussions, and the Deutsche Forschungsgemeinschaft (DFG) for funding of my position by the Heisenberg Programme (LA 2984/3-1).

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Authors and Affiliations

  1. Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

    Michael Lammers

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  1. Michael Lammers
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Correspondence to Michael Lammers .

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Editors and Affiliations

  1. Structural and Computational Biology Unit & Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany

    Edward A. Lemke

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Lammers, M. (2018). Expression and Purification of Site-Specifically Lysine-Acetylated and Natively-Folded Proteins for Biophysical Investigations. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_11

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  • DOI: https://doi.org/10.1007/978-1-4939-7574-7_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7573-0

  • Online ISBN: 978-1-4939-7574-7

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