6-羟基烟酸3-单加氧酶NicC的纯化与结晶

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

【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nic C,纯化重组Nic C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p ET28a-nic C,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p ET28a-nic C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:0.2mol/LNH4Cl,0.01mol/LCaCl2·2H2O,31%PEG4000,0.05mol/LTris-HClp H8.0,4°C;Se Met-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。

[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 nic C was cloned and expressed in Escherichia coli,the NicC protein was purified and the crystallization conditions were studied.[Methods]The nic C gene was amplified from the genomic DNA of Pseudomonas putida KT2440 and a recombinant expression vector p ET28 a-nic C 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 p ET28 a-nic C 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 NH4 Cl,0.01 mol/L Ca Cl2·2 H2 O,31%PEG4000,0.05 mol/L Tris-HCl p H 8.0,4°C for NicC,and 0.2 mol/L NH4 Cl,0.01 mol/L Ca Cl2·2 H2 O,0.05 mol/L Tris-HCl p H 7.9,31%PEG3350,4°C for Se Met-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 toβ-position monooxygenase to recognize the pyridine ring-containing substrate and catalyze the hydroxylation ofβ-position on the pyridine ring of the substrate.