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Applied Biochemistry and Biotechnology

, Volume 163, Issue 1, pp 71–79 | Cite as

Efficient Enzymatic Production of the Bacterial Second Messenger c-di-GMP by the Diguanylate Cyclase YdeH from E. coli

  • Franziska Zähringer
  • Claudia Massa
  • Tilman SchirmerEmail author
Article

Abstract

Cyclic di-GMP (c-di-GMP) is an almost universal bacterial second messenger involved in the regulation of cell surface-associated traits and the persistence of infections. GGDEF and EAL domain-containing proteins catalyse c-di-GMP synthesis and degradation, respectively. We report the enzymatic large-scale synthesis of c-di-GMP, making use of the GGDEF domain-containing protein YdeH from Escherichia coli. Overexpression and purification of YdeH have been established, and the conditions for c-di-GMP synthesis were optimised. In contrast to the chemical synthesis of c-di-GMP, enzymatic c-di-GMP production is a one-step reaction that can easily be performed with the equipment of a standard biochemical lab. The protocol allows the production of milligram amounts of c-di-GMP within 1 day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processes.

Keywords

c-di-GMP Diguanylate cyclase GGDEF domain Enzymatic synthesis E. coli 

Notes

Acknowledgement

We thank Dr. Alexander Böhm, Biozentrum Basel, for the gift of the plasmid, Dr. Martin Allan, Biozentrum Basel, for help with 1H-NMR spectroscopy and Dietrich Samoray for suggesting to test the effect of osmolytes on YdeH solubility. The work was supported by Swiss National Science Foundation grant 3100A0-105587.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Franziska Zähringer
    • 1
  • Claudia Massa
    • 1
  • Tilman Schirmer
    • 1
    Email author
  1. 1.Core Program Structural Biology and Biophysics, BiozentrumUniversity of BaselBaselSwitzerland

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