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

Content Provider	Springer Nature Link
Author	Spehr, Volker Warrass, Ralf Höcherl, Klaus Ilg, Thomas
Copyright Year	2011
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.
Starting Page	761
Ending Page	775
Page Count	15
File Format	PDF
ISSN	02732289
Journal	Applied Biochemistry and Biotechnology
Volume Number	165
Issue Number	3-4
e-ISSN	15590291
Language	English
Publisher	Humana Press Inc
Publisher Date	2011-06-28
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	C-di-GMP Diguanylate cyclase Enzymatic cascade Immunomodulation Biotechnological production Biochemistry Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Environmental Engineering Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

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