假坚强芽孢杆菌四氢嘧啶羟化酶的晶体制备及X-射线衍射研究

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

摘要:

摘要:【目的】假坚强芽孢杆菌四氢嘧啶羟化酶蛋白纯化、晶体制备及X-射线衍射研究。【方法】通过PCR从假坚强芽孢杆菌OF4中克隆获得四氢嘧啶羟化酶基因，构建原核表达载体，经过原核表达，采用Ni-NTA亲和层析法和分子排阻色谱法纯化蛋白，289 K下采用座滴法进行晶体筛选和制备，在低温100 K下通过X-射线衍射仪(Rigaku MicroMax-007 HF)收集晶体衍射数据。【结果】通过原核表达及纯化成功获得了适合晶体生长的蛋白BpEctD。通过筛选最终在蛋白浓度为6.5 mg/mL及含有0.2 mol/L MgCl2·6H2O，0.1 mol/L Bis-Tris pH6.5，25% (W/V) 聚乙二醇3，350的缓冲液中获得了理想的蛋白晶体，其大小约为360 μm×240μm×60 μm，并在100K下成功收集了衍射数据，晶体衍射分辨率为2.40，空间群为三斜晶系P1，晶胞参数为a=45.18，b=58.87，c=68.81，α=77.48°，β=86.03°，γ=66.97°，每个不对称单位中含有2 个BpEctD单体，马修斯系数为2.44 3/Da，溶剂含量约为49.53%。【结论】衍射数据的成功收集为假坚强芽孢杆菌OF4四氢嘧啶羟化酶三维结构的解析奠定了前期基础，将有助于阐明四氢嘧啶羟化酶的催化机制。

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.