首页> 美国卫生研究院文献>Acta Crystallographica Section F: Structural Biology and Crystallization Communications >Protein purification crystallization and preliminary X-ray diffraction analysis of l-arabinose isomerase from Lactobacillus fermentum CGMCC2921
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Protein purification crystallization and preliminary X-ray diffraction analysis of l-arabinose isomerase from Lactobacillus fermentum CGMCC2921

机译:发酵乳杆菌CGMCC2921的1-阿拉伯糖异构酶的蛋白质纯化结晶和X射线初步分析

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摘要

l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose, as well as that of d-galactose to d-tagatose. A thermophilic AI derived from Lactobacillus fermentum CGMCC2921 (LFAI) was overexpressed in Escherichia coli BL21 (DE3). This enzyme was purified to over 95% purity by nickel affinity, Mono-Q ion-exchange and size-exclusion chromatography. The LFAI protein was crystallized from either 0.1 M bis-tris pH 6.5, 23% PEG 3350, 0.3 M NaCl (form 1 crystals) or 0.1 M bis-tris pH 6.0, 25% PEG monomethyl ether 5000 (form 2 crystals). Diffraction data from form 1 LFAI crystals were collected to 2.80 Å resolution using synchrotron radiation. The form 1 crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.11, b = 184.57, c = 186.26 Å, α = β = γ = 90°. The asymmetric unit contained six LFAI subunits, corresponding to a calculated Matthews coefficient of 2.29 Å3 Da−1 and a solvent content of 46.22%.
机译:1-阿拉伯糖异构酶(AI)催化1-阿拉伯糖异构化为1-核糖,以及将d-半乳糖异构化为d-塔格糖。源自发酵乳杆菌CGMCC2921(LFAI)的嗜热性AI在大肠杆菌BL21(DE3)中过表达。通过镍亲和力,Mono-Q离子交换和尺寸排阻色谱法将该酶纯化至纯度超过95%。 LFAI蛋白从0.1 M bis-tris pH 6.5、23%PEG 3350、0.3 M NaCl(形式1晶体)或0.1 M bis-tris pH 6.0、25%PEG单甲基醚5000(形式2晶体)中结晶。使用同步加速器辐射从形式1 LFAI晶体收集的衍射数据达到2.80Å分辨率。 1型晶体属于正交晶空间群P212121,单位晶胞参数a = 85.11,b = 184.57,c = 186.26Å,α=β=γ= 90°。不对称单元包含六个LFAI亚基,对应于2.29Å 3 Da -1 的马修斯系数和46.22%的溶剂含量。

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