Spehr, Volker and Warrass, Ralf and Hoecherl, Klaus and Ilg, Thomas (2011) Large-Scale Production of the Immunomodulator c-di-GMP from GMP and ATP by an Enzymatic Cascade. APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 165 (3-4). pp. 761-775. ISSN 0273-2289,
Full text not available from this repository. (Request a copy)Abstract
(3'aEuro"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 1 beta, tumor necrosis factor alpha, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | 3',5'-CYCLIC DIGUANYLIC ACID; POLYMERASE CHAIN-REACTION; ACETOBACTER-XYLINUM; CELLULOSE SYNTHESIS; CYCLIC DIGUANYLATE; PSEUDOMONAS-AERUGINOSA; SIGNALING MOLECULE; ALLOSTERIC CONTROL; BIOFILM FORMATION; VACCINE ADJUVANT; C-di-GMP; Diguanylate cyclase; Enzymatic cascade; Immunomodulation; Biotechnological production |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Physiologie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 28 May 2020 07:19 |
| Last Modified: | 28 May 2020 07:19 |
| URI: | https://pred.uni-regensburg.de/id/eprint/20022 |
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