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Updated: May 11, 2026
Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Cloning, expression, purification, crystallization and preliminary X-ray analysis of EaLsc, a levansucrase from
Lorenzo Caputi1, Michele Cianci, Stefano Benini
1Laboratory of Bioorganic Chemistry and Crystallography, Faculty of Science and Technology, Free University of Bolzano, Piazza Università 5, 39100 Bolzano, Italy.
Abstract:
The Gram-negative bacterium Erwinia amylovora is a destructive pathogen of Rosaceae. During infection, E. amylovora produces the exopolysaccharide levan, which contributes to the occlusion of plant vessels, causing the wilting of shoots. Levan is a fructose polymer that is synthesized by multifunctional enzymes called levansucrases. The levansucrase from E. amylovora (EaLsc) was heterologously expressed as a GST-fusion protein in Escherichia coli, purified and crystallized after tag removal. The protein crystallized in space group P21212. X-ray diffraction data were acquired to 2.77 Å resolution. The structure of the enzyme was solved by molecular replacement. The asymmetric unit contains eight enzyme molecules, giving a solvent content of 58.74% and a Matthews coefficient of 2.98 Å(3) Da(-1).
