Crystallization and X-ray diffraction of an ectoine hydroxylase from Bacillus pseudofirmus OF4

Abstract:

Abstract:［Objective］To study crystallization and X-Ｒay diffraction of ectoine hydroxylase from Bacillus pseudofirmus OF4.［Methods］We cloned the gene BpectD from B．pseudofirmus OF4 with a His6 tag and overexpressed it in E．coli BL21(DE3)． Then，we purified protein BpEctD by Ni2 + -chelating affinity and size-exclusion chromatography．After that，crystals were grown by the sitting-drop vapour-diffusion method at 289 K and diffracted at 100K using an in-house X-ray source．［Results］Protein BpEctD was expressed and purified successfully． We obtained well diffracting crystals of about 360 μm×240 μm×60 μm in size using a solution consisting of 0.2 mol/L magnesium chloride hexahydrate，0.1 mol /L bis-tris pH6.5，25% (W/V) polyethylene glycol 3，350 at a protein concentration of 6.5 mg/mL，and collected X-ray diffraction data to 2.40  resolution in the anorthic space group P1，with unit-cell parameters a=45.18，b=58.87，c=68.81，α=77.48°，β=86.03°，γ=66.97°．The asymmetric unit contains two molecules of BpEctD with a Mattews coefficient of about 2.443/Da and a solvent content of 49.53%．［Conclusion］ According to the X-ray diffraction data，the three-dimensional structure of BpEctD from B．pseudofirmus OF4 soon will be analyzed，and it will provide insights into the biochemical properties of ectoine hydroxylase.