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- Title
Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase.
- Authors
Magellan, Hervé; Drujon, Thierry; Thellend, Annie; Piffeteau, Annie; Becker, Hubert F.
- 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.
- Subjects
GENE expression; FILAMENTOUS fungi; BOTRYTIS cinerea; CHITIN; POLYSACCHARIDES; MICROFUNGI; GLYCOSYLTRANSFERASES; PROTEINS; ESCHERICHIA
- Publication
BMC Research Notes, 2010, Vol 3, p299
- ISSN
1756-0500
- Publication type
Article
- DOI
10.1186/1756-0500-3-299