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Enzyme-Mediated Ligation Methods pp 279–297Cite as

Lipoic Acid Ligase-Promoted Bioorthogonal Protein Modification and Immobilization

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

Abstract

Protein bioconjugation benefits from precise regional and temporal control. One notable way of achieving this control is through the enzymatic attachment of bioorthogonal reactive handles to peptide recognition sequences that are genetically fused to target proteins of interest. The lipoic acid ligase variant, LplAW37V, functionalizes proteins by covalently attaching an azide-bearing lipoic acid derivative to a 13-amino acid recognition sequence known as the lipoic acid ligase acceptor peptide (LAP). Once attached, the azide group can be modified with diverse chemical entities through azide–alkyne click chemistry, enabling conjugation of chemical probes such as fluorophores and facilitating polymer attachment, glycosylation, and protein immobilization in addition to many other possible chemical modifications. The versatility of the attached azide group is complemented by the modular nature of the LAP sequence, which can be introduced within a protein at internal and/or terminal sites as well as at multiple sites simultaneously. In this chapter we describe the in vitro LplAW37V-mediated ligation of 10-azidodecanoic acid to a LAP-containing target protein (i.e., green fluorescent protein (GFP)) and the characterization of the ligation reaction products. Additionally, methods for the modification and immobilization of azide-functionalized LAP-GFP are discussed.

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Acknowledgments

This work was supported by the Natick Soldier Research, Development, and Engineering Center (NSRDEC) through a research grant (W911NF-12-01115) awarded by the Army Research Office.

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA

    Joseph G. Plaks & Joel L. Kaar

Authors
  1. Joseph G. Plaks
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  2. Joel L. Kaar
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Corresponding author

Correspondence to Joel L. Kaar .

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Editors and Affiliations

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

    Timo Nuijens  & Marcel Schmidt  & 

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Plaks, J.G., Kaar, J.L. (2019). Lipoic Acid Ligase-Promoted Bioorthogonal Protein Modification and Immobilization. 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_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9546-2_14

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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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