Cloning, expression, purification, crystallization and preliminary X-ray analysis of EaLsc, a levansucrase from Erwinia amylovora

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).

Keywords: Erwinia; fructooligosaccharides; fructosyltransferases; levan; levansucrases.

Figure 1

Purification of EaLsc. (a) Elution profile of EaLsc on size-exclusion chromatography (SEC) using a Superdex 75 16/60 column. (b) SDS–PAGE analysis of peak fractions from SEC purification, showing the homogeneity of EaLsc immediately prior to crystallization.

Figure 2

A cluster of EaLsc crystal plates obtained by hanging-drop vapour diffusion by adding 1 µl 35% PEG 2000 MME and 0.1 M KSCN to 1 µl protein solution followed by equilibration against 1 ml 35% PEG 2000 MME, 0.1 M KSCN.

Figure 3

A diffraction image of an EaLsc crystal collected on a PILATUS 6M detector.