Highly Precise Protein Semisynthesis through Ligation–Desulfurization Chemistry in Combination with Phenacyl Protection of Native Cysteines

  • Somnath Mukherjee
  • Maria Matveenko
  • Christian F. W. BeckerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2133)


Semisynthesis of proteins via expressed protein ligation is a powerful tool to furnish full-length proteins carrying site-specific (posttranslational) modifications. The development of various β-mercapto amino acid building blocks coupled with ligation–desulfurization chemistry enabled further advances in this methodology by alleviating the need for cysteine residues at the desired ligation sites. However, this expansion in the availability of viable ligation sites is sometimes counterbalanced by the inadvertent desulfurization of unprotected native cysteines, which might be of structural and/or functional importance. Here, we provide a detailed protocol for using the cysteine-selective protecting group phenacyl (PAc) to achieve precise protein semisynthesis preserving native cysteine residues. The PAc group can be easily installed on cysteine(s) within recombinantly produced protein thioesters, withstands standard ligation, desulfurization and reversed phase HPLC conditions, and can be smoothly removed. We have previously demonstrated the utility of this protecting group through the semisynthesis of two model proteins, human small heat shock protein Hsp27 and Prion protein, in which one or two native cysteines, respectively, were maintained through the ligation–desulfurization sequence.

Key words

Posttranslational modifications Protein protecting group Cysteine-selective protection Phenacyl Expressed protein ligation Ligation–desulfurization Radical desulfurization Traceless protein semisynthesis 



This work was supported by the Austrian Academy of Sciences by an APART fellowship to M.M.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Somnath Mukherjee
  • Maria Matveenko
  • Christian F. W. Becker
    • 1
    Email author
  1. 1.Institute of Biological Chemistry, Faculty of ChemistryUniversity of ViennaViennaAustria

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