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Large-Scale Production of the Immunomodulator c-di-GMP from GMP and ATP by an Enzymatic Cascade

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Abstract

(3′–5′)-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a “one-pot” three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1β, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.

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Acknowledgments

We thank Nicole Engelmann, Karl-Heinz Grimm, Susanne Schmalz, and Heinz-Jörg Wennesheimer for expert technical assistance.

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Authors and Affiliations

  1. Intervet Innovation GmbH, Zur Propstei, 55270, Schwabenheim, Germany

    Volker Spehr, Ralf Warrass & Thomas Ilg

  2. Institut für Physiologie, Universität Regensburg, 93053, Regensburg, Germany

    Klaus Höcherl

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  1. Volker Spehr
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Correspondence to Thomas Ilg.

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Spehr, V., Warrass, R., Höcherl, K. et al. Large-Scale Production of the Immunomodulator c-di-GMP from GMP and ATP by an Enzymatic Cascade. Appl Biochem Biotechnol 165, 761–775 (2011). https://doi.org/10.1007/s12010-011-9294-z

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