6-羟基-3-琥珀酰吡啶单加氧酶的纯化与结晶条件

Purification and crystallization of 6-hydroxy-3-succinoyl-pyridine monooxygenase

摘要:

【目的】在大肠杆菌中克隆表达尼古丁降解关键的6-羟基-3-琥珀酰吡啶单加氧酶基因hspB，纯化重组HspB蛋白并进行结晶条件的初步研究。【方法】从恶臭假单胞菌S16基因组中PCR扩增hspB基因，构建重组表达载体pET28a-hspB，并在E. 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HspB蛋白进行结晶条件筛选和优化。【结果】本文成功构建重组质粒pET28a-hspB并纯化获得达到结晶纯度的HspB蛋白。结晶条件初筛和优化后获得可培养HspB蛋白晶体的条件为0.2 mol/L NaCl、0.1 mol/L HEPES pH 7.5、1.1 mol/L (NH4)2SO4、4 °C、加晶种。【结论】HspB蛋白纯化体系的构建和结晶条件的初步研究为从结构生物学的角度进一步研究HspB结构与功能的关系、定向进化提高HspB催化效率奠定了基础。

Abstract:

[Objective] hspB, the gene of the key monooxygenase in nicotine degradation from Pseudomonas putida S16, was cloned and expressed in Escherichia coli; protein HspB was purified, and the crystal condition of HspB was studied. [Methods] The gene hspB was amplified from the genomic DNA of Pseudomonas putida S16. Then recombination plasmid pET28a-hspB was expressed in E. coli BL21. Ni2+-NTA His·Bind and gel filtration were used to purify HspB. The preliminary crystal of HspB was screened and optimized by using hanging drop diffusion method. [Results] pET28a-hspB plasmid was successfully constructed and the protein HspB was purified to crystalline purity. Crystal condition of HspB was obtained and the culture condition is 0.2 mol/L NaCl, 0.1 mol/L HEPES pH 7.5, 1.1 mol/L (NH4)2SO4, 4 °C, Seeding. [Conclusion] The construction of the HspB purification system and the study of preliminary crystal of HspB lay a foundation for the research of structure-function relationship and improvement of HspB catalytic efficiency by directed evolution of gene manipulation.