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Trypsiligase-Catalyzed Peptide and Protein Ligation

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Enzyme-Mediated Ligation Methods

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2012))

Abstract

Site-specific incorporation of nonproteinogenic functionalities into protein targets is an important tool in both basic and applied research and represents a major challenge to protein chemists. Chemical labeling methods often target multiple positions within a protein and therefore suffer from a lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this chapter we describe the application of the highly specific trypsin variant trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Reactions rapidly proceed under mild conditions and result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the synthesis of peptide (ester) substrates. In addition, we provide instructions for the bioconjugation reactions and for the qualitative and quantitative analysis of reaction progress and efficiency.

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Authors and Affiliations

  1. Institute of Biochemistry/Biotechnology, Charles-Tanford-Protein Center, Martin-Luther-University Halle-Wittenberg, Halle, Germany

    Sandra Liebscher & Frank Bordusa

Authors
  1. Sandra Liebscher
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  2. Frank Bordusa
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Corresponding author

Correspondence to Frank Bordusa .

Editor information

Editors and Affiliations

  1. EnzyPep B.V., Geleen, The Netherlands

    Timo Nuijens  & Marcel Schmidt  & 

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Liebscher, S., Bordusa, F. (2019). Trypsiligase-Catalyzed Peptide and Protein Ligation. In: Nuijens, T., Schmidt, M. (eds) Enzyme-Mediated Ligation Methods. Methods in Molecular Biology, vol 2012. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9546-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-9546-2_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9545-5

  • Online ISBN: 978-1-4939-9546-2

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