title: Large-scale production of the immunomodulator c-di-GMP from GMP and ATP by an enzymatic cascade.
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.
authors.affiliation: Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Spehr
authors.forename: Ralf
authors.initials: R
authors.lastname: Warrass
authors.forename: Klaus
authors.initials: K
authors.lastname: Höcherl
authors.forename: Thomas
authors.initials: T
authors.lastname: Ilg
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000494:pharmacology; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism*; D051528:Guanylate Kinases / Q000235:genetics / Q000378:metabolism*; D007155:Immunologic Factors / Q000096:biosynthesis* / Q000494:pharmacology; D053583:Interleukin-1beta / Q000096:biosynthesis / Q000276:immunology; D015850:Interleukin-6 / Q000096:biosynthesis / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000276:immunology
chemical_list: D029968:Escherichia coli Proteins; D007155:Immunologic Factors; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C062025:bis(3',5')-cyclic diguanylic acid; D006157:Guanosine Monophosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D051528:Guanylate Kinases; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP