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目的 AphB是霍乱弧菌毒力基因表达调控网络中重要的转录调控因子,可激活毒力基因表达,本实验目的在于得到AphB蛋白C端结构域的晶体结构,为研究其与辅助诱导物结合之后的构象改变对于毒力调控网络的影响奠定基础。方法以霍乱弧菌的AphB蛋白C端结构域为对象,将目的基因克隆到表达载体pET-32a(+)上,使重组质粒在大肠埃希菌中高效表达目的蛋白,然后利用镍离子亲和层析、MonoQ离子交换层析和GF75凝胶过滤层析法进行纯化。分别选取5、10、20和30mg/ml的目的蛋白,采用悬滴气相扩散法进行蛋白结晶,筛选结晶试剂盒条件,在得到初晶的结晶条件基础上通过改变蛋白与池液混合比例、结晶池液成分、环境温度等进行结晶条件的优化。结果含有目的基因的重组质粒在大肠埃希菌中高效表达可溶性目的蛋白。利用3种蛋白纯化方法依次进行纯化,得到较高纯度的目的蛋白。对蛋白结晶试剂盒结晶条件进行优化,得到的最优结晶条件为:蛋白浓度为30mg/ml;池液成分为0.1mol/L BIS-TRIS,2.0mol/L Ammonium sulfate,pH 5.0,18℃环境孵育24h。用优化的结晶条件进行结晶处理,得到分辨率为2.1的蛋白晶体。结论通过原核表达和用优化的结晶条件得到了霍乱弧菌AphB蛋白C端结构域的蛋白晶体,所得数据为蛋白的空间结构解析提供了重要依据,对蛋白C端结构域与辅助诱导物结合的功能研究奠定了基础。
Objective AphB is an important transcriptional regulatory factor in the Vibrio cholerae virulence gene expression regulatory network, which can activate virulence gene expression. The purpose of this experiment is to obtain the crystal structure of C-terminal domain of AphB protein. The conformational changes laid the foundation for the influence of virulence regulation network. Methods The Vibrio cholerae C-terminal domain of AphB protein was cloned into the expression vector pET-32a (+). The recombinant plasmid was then used to express the target protein in Escherichia coli. Then, Chromatography, MonoQ ion exchange chromatography, and GF 75 gel filtration chromatography. The target proteins of 5, 10, 20 and 30 mg / ml were respectively selected, and the protein was crystallized by the hanging drop gas diffusion method. The conditions of the crystallization kit were selected. Based on the crystallization conditions of the primary crystals, Liquid pool composition, ambient temperature, etc. to optimize the crystallization conditions. As a result, the recombinant plasmid containing the target gene highly expressed the soluble protein of interest in Escherichia coli. Using three kinds of protein purification methods were purified in order to obtain a higher purity of the target protein. The crystallization conditions of the protein crystallization kit were optimized. The optimal crystallization conditions were as follows: the protein concentration was 30mg / ml; the pool composition was 0.1mol / L BIS-TRIS, 2.0mol / L Ammonium sulfate, Incubate 24h. Crystallization with optimized crystallization conditions gave a protein crystal with a resolution of 2.1 . Conclusion The protein crystal of C-terminal domain of Vibrio cholerae AphB protein was obtained by prokaryotic expression and optimized crystallization conditions. The data provided an important basis for the analysis of the spatial structure of the protein. The binding of C-terminal domain to the accessory inducer Functional research laid the foundation.