Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase. - Archive ouverte HAL Access content directly
Journal Articles BMC Research Notes Year : 2010

Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase.

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Abstract

BACKGROUND: Chitin synthase 3a (CHS3a) from Botrytis cinerea (Bc) catalyses the multiple transfer of N-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a β-1,4 linked GlcNAc homopolymer, is an essential cell wall component of filamentous fungi. Chitin synthase, processive membranous protein, has been recognized as a promising target for new antifungicides. Enzymatic characterizations of chitin synthases have been limited, mainly because purity and amounts of integral enzyme obtained after purification procedures have not been sufficient. FINDINGS: We undertook the preparation of two BcCHS3a fragment proteins, containing only the central domain and devoid of the N-terminal and transmembrane C-terminal regions. The central domain of CHS3a, named SGC (Spsa GntI Core), is conserved in all UDP-glycosyltransferases and it is believed to contain the active site of the enzyme. CHS3a-SGC protein was totally expressed as inclusion bodies in Escherichia coli. We performed recombinant CHS3a-SGC purification in denaturing conditions, followed by a refolding step. Although circular dichroism spectra clearly exhibited secondary structures of renatured CHS3a-SGC, no chitin synthase activity was detected. Nevertheless CHS3a-SGC proteins show specific binding for the substrate UDP-GlcNAc with a dissociation constant similar to the Michaelis constant and a major contribution of the uracil moiety for recognition was confirmed. CONCLUSIONS: Milligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be easily obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a.
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inserm-00663713 , version 1 (27-01-2012)

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Hervé Magellan, Thierry Drujon, Annie Thellend, Annie Piffeteau, Hubert F. Becker. Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase.. BMC Research Notes, 2010, 3 (1), pp.299. ⟨10.1186/1756-0500-3-299⟩. ⟨inserm-00663713⟩
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