中文摘要：

应用激光镊子拉曼光谱系统（LTRS）,实时分析不同温度下蚕豆14-3-3b可溶性蛋白和包涵体蛋白在重组大肠杆菌细胞中的动态表达水平。结果显示,14-3-3b可溶性蛋白与包涵体蛋白有明显不同的拉曼光谱特征峰,反映出两者在主链和侧链构象上的差异性;在28℃时,包涵体蛋白特征峰900,1033,1328,1415和1446cm^-1强度随着诱导时间延长而增加的幅度,明显大于可溶性蛋白的特征峰763,1002,1363,1451和1665cm-1的增加幅度,16℃时诱导表达结果正好相反,可溶性蛋白的特征峰增强幅度显著大于包涵体蛋白,说明28℃诱导培养条件下,重组菌蛋白质过量表达以形成包涵体为主,16℃较低温度条件下以形成可溶性蛋白为主,正确折叠蛋白增加的信息可以通过光谱变化反映出来,在该温度下,蛋白质的正确折叠有利于细胞形成稳定的可溶性蛋白;另外,重组菌在相对低温条件下更多地表达含胱氨酸的非重组蛋白,可能与蛋白质折叠相关。LTRS技术可以在单个细胞水平上对大肠杆菌细胞过量表达可溶性蛋白和包涵体蛋白过程进行无损害、无标记的实时分析。

英文摘要：

Laser tweezers Raman spectroscopy （LTRS） is used for real-time investigation of the overexpression of broad bean 14-3-3b soluble protein and inclusion body protein in the single living Escherichia coli cell at different temperatures after induction with isopropyl thiogalactoside （IPTG）. The results show that 14-3-3b soluble protein and inclusion body protein have significantly different Raman characteristic peaks, which reflects that the two kinds of proteins have distinct conformations in their main chains and side chains. Intensity of the characteristic peaks of the inclusion body protein, including 900, 1033, 1328, 1415 and 1446 cm^-1 , gradually increases as the function of the culture time after IPTG is added at 28 ℃, however, intensity of the peaks of the soluble protein, including 763, 1002, 1363, 1451 and 1665 cm^-1 , increases in small increments relatively. Conversely, the growth rate of soluble protein peaks is higher than that of the inclusion body peaks obviously at 16 ℃ , indicating that the recombinant protein is mainly formed as inclusion body at 28 ℃, and mainly as soluble protein at 16℃. Under lower temperature, the information of protein folding in precision can be reflected by the spectral changes and the correct protein folding into certain configuration is conducive to the formation of soluble protein in E. eoli cells. In addition, the non-recombinant protein of the bacteria expression at 16 ℃ contains more cystine, which may be associated with protein folding. The LTRS is a useful tool for nondestructive, unmarked, efficient, real-time monitoring of overexpression processes of soluble protein and inclusion body protein in a single living E. eoli cell.