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Updated: May 28, 2026

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile
Published on: November 3, 2018
The CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity
Michaël Kupper1, Cédric Bauvois, Jean-Marie Frère
1Institute of Molecular Biotechnology, RWTH-Aachen University, Forckenbeckstraße 6, Aachen, Germany.
Abstract:
The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold.
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