Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase
1 UPMC Univ Paris 06, CNRS UMR 7203, Laboratoire des Biomolécules, Paris F-75005, France
2 et Université Paris Diderot, Paris, France
3 Laboratoire d'Optique et Biosciences, INSERM U696, CNRS UMR7645, Ecole Polytechnique, Palaiseau, France
BMC Research Notes 2010, 3:299 doi:10.1186/1756-0500-3-299Published: 11 November 2010
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.
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 (
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.