13C6-酪氨酸标记的蛋清溶菌酶的表达及NMR测定

Expression of 13C6-Tyrosine-Labeled Hen-Egg-White Lysozyme in E. Coli and Its NMR Determination

针对蛋白质核磁共振(NMR)信号复杂难以解析的问题,选用选择性标记方法可简化蛋白质NMR谱图。本实验以蛋清溶菌酶(HEL)为目标蛋白,构建了pET-HEL蛋白表达载体,转入到大肠杆菌Ε.coliBL21(DE3)中,得到重组菌株。然后通过改变诱导温度、诱导浓度以及诱导时间等条件来优化HEL在大肠杆菌Ε.coliBL21(DE3)中的表达条件,通过SDS-PAGE电泳分析,确定了最佳表达条件:37℃、0.4mmol/LIPTG诱导8h。按最佳条件于诱导前加入13C6-酪氨酸进行选择性标记,扩大表达,经过变复性处理与Ni柱纯化后,获得选择性标记的HEL。进一步制备标记蛋白核磁样品进行测定,得到简化的NMR谱。本研究为采用NMR简化分析蛋白质的结构奠定了基础。

Protein nuclear magnetic resonance (NMR) signals are complex and they are difficult to analyze, and a selective labeling method can be used to simplify the protein NMR spectrum. In this paper, hen egg white lysozyme (HEL) is used as the target protein. The pET-HEL protein expression vector is constructed and transferred into E.coli BL21 (DE3) to obtain a recombinant strain. The optimium expression conditions of HEL in E.coli BL21(DE3) are as followes: the inducing temperature 37℃, concentration of inducer 0.4 mmol/L IPTG, and inducing time 8 h. Under the optimum conditions, 13C6-tyrosine is added for selective labeling to express. After refolding and the purification of the Ni column, the target protein is obtained. Further, a nuclear magnetic sample is prepared and measured to get a simplified NMR spectrum. It lays the foundation for the usage of NMR to simplify the analysis of protein structure.