Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein

  • Bruno Fauvet
  • Hilal A. Lashuel
Part of the Methods in Molecular Biology book series (MIMB, volume 1345)


Posttranslational modifications (PTMs) serve as molecular switches for regulating protein folding, function, and interactome and have been implicated in the misfolding and amyloid formation by several proteins linked to neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Understanding the role of individual PTMs in protein misfolding and aggregation requires the preparation of site-specifically modified proteins, as well as the identification of the enzymes involved in regulating these PTMs. Recently, our group has pioneered the development of enzymatic, synthetic, and semisynthetic strategies that allow site-specific introduction of PTMs at single or multiple sites and generation of modified proteins in milligram quantities. In this chapter, we provide detailed description of enzymatic and semisynthetic strategies for the generation of the phosphorylated α-Synuclein (α-Syn) at S129, (pS129), which has been identified as a pathological hallmark of Parkinson’s disease. The semisynthetic method described for generation of α-Syn-pS129 requires expertise with protein chemical ligation, but can be used to incorporate other PTMs (single or multiple) within the α-Syn C-terminus if desired. On the other hand, the in vitro kinase-mediated phosphorylation strategy does not require any special setup and is rather easy to apply, but its application is restricted to the generation of α-Syn_pS129. These methods have the potential to increase the availability of pure and homogenous modified α-Syn reagents, which may be used as standards in numerous applications, including the search for potential biomarkers of synucleinopathies.

Key words

Parkinson’s disease Posttranslation modification Alpha-synuclein Amyloid Phosphorylation Semisynthesis Native chemical ligation Desulfurization 



This work has been possible thanks to the tremendous efforts of all the members of the Lashuel group that have contributed to developing and optimizing the protocols described here. We wish to specially thank Mr. John Perrin for the enzymatic preparation of α-Syn_pS129; as well as Mr. Anass Chiki, Dr. Sean Deguire, and Dr. John Warner for helpful comments on the manuscript. This work was supported by grants from the European Research Council (ERC grant n° 243182), the Michael J. Fox Foundation for Parkinson’s Research (grant n° 531107), and Ecole Polytechnique Fédérale de Lausanne Swiss National Science Foundation (grant n (31003A_120653).


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind InstituteEcole Polytechnique Fédérale de LausanneLausanneSwitzerland
  2. 2.Qatar Biomedical Research InstituteHamad Bin Khalifa UniversityDohaQatar

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