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6-羟基烟酸3-单加氧酶Ni cC的纯化与结晶
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国家自然科学基金(31670106)


Purification and crystallization of 6-hydroxynicotinic acid 3-monooxygenase NicC
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    摘要:

    【背景】含氮杂环吡啶化合物是重要的环境污染物之一,在人体内长期积累会导致肿瘤/畸胎等疾病,利用微生物降解含氮杂环化合物是一种非常有效的途径。【目的】在大肠杆菌中克隆表达6-羟基烟酸3-单加氧酶基因nicC,纯化重组NicC蛋白并进行结晶条件的研究。【方法】以恶臭假单胞菌KT2440为模板对nicC基因进行PCR扩增,构建重组表达载体pET28a-nicC,在大肠杆菌(Escherichia coli) BL21(DE3)中诱导表达,利用亲和层析和凝胶过滤层析纯化重组蛋白。利用悬滴扩散法对NicC蛋白进行结晶条件筛选和优化。【结果】成功构建重组质粒pET28a-nicC并纯化获得达到结晶纯度的NicC蛋白。通过结晶条件初筛和正交优化实验发现,NicC蛋白的最佳结晶条件为:0.2 mol/L NH4Cl,0.01 mol/L CaCl2·2H2O,31% PEG4000,0.05 mol/L Tris-HCl ph 8.0,4 °C;SeMet-NicC蛋白和NicC与6-HNA共晶的最佳结晶条件为:0.2 mol/L NH4Cl,0.01 mol/L CaCl2·2H2O,0.05 mol/L Tris-HCl ph 7.9,31% PEG3350,4 °C。【结论】NicC蛋白纯化体系的构建和结晶条件的研究为最终解析NicC蛋白三维结构提供了有利条件,为揭示吡啶环β位单加氧酶识别含吡啶环底物并催化底物的吡啶环上β位羟基化的分子机理奠定了基础。

    Abstract:

    [Background] N-heterocyclic aromatic compounds (NHACs) are one of the important environmental pollutants. Long-term accumulation in the human body can lead to diseases. The degradation of N-heterocyclic aromatic compounds by microorganism is an effective approach. [Objective] The 6-hydroxynicotinic acid 3-monooxygenase gene nicC was cloned and expressed in Escherichia coli, the NicC protein was purified and the crystallization conditions were studied. [Methods] The nicC gene was amplified from the genomic DNA of Pseudomonas putida KT2440 and a recombinant expression vector pET28a-nicC was constructed and expressed in E. coli BL21(DE3). Affinity and gel filtration chromatography were used to purify NicC. The preliminary screening and optimization of crystals were done by using hanging drop diffusion method. [Results] The pET28a-nicC was constructed successfully and the purified NicC protein was obtained. Through preliminary screening of crystallization conditions and orthogonal optimization experiments, the optimal crystallization conditions were 0.2 mol/L NH4Cl, 0.01 mol/L CaCl2·2H2O, 31% PEG4000, 0.05 mol/L Tris-HCl ph 8.0, 4 °C for NicC, and 0.2 mol/L NH4Cl, 0.01 mol/L CaCl2·2H2O, 0.05 mol/L Tris-HCl ph 7.9, 31% PEG3350, 4 °C for SeMet-NicC and cocrystals of NicC and 6-HNA. [Conclusion] The construction of NicC protein purification system and study of crystallization conditions provided favorable conditions for the final analysis of the three-dimensional structure of NicC protein. The results laid a foundation for revealing the molecular mechanism of the pyridine ring to β-position monooxygenase to recognize the pyridine ring-containing substrate and catalyze the hydroxylation of β-position on the pyridine ring of the substrate.

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郭丽华,唐鸿志,吴更. 6-羟基烟酸3-单加氧酶Ni cC的纯化与结晶[J]. 微生物学通报, 2020, 47(7): 2021-2027

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  • 在线发布日期: 2020-07-06
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