【摘要】 采用傅里叶变换显微红外手段研究了在信号肽缺失的情况下,C-末端酸性蛋白的存在对小麦α-硫素原核表达过程中蛋白质二级结构的影响。SDS-PAGE表明带有酸性蛋白(Sc样品)的重组质粒在大肠杆菌BL21(DE3)中以包涵体形式高效表达;而酸性蛋白缺失(S样品)可以极大提高外源蛋白的可溶性。通过对含有S及Sc的全细胞在诱导前及诱导后2h的酰胺Ⅰ带区域图谱求差谱及二阶导数谱,发现诱导前在1630cm-1处吸收较弱,而在诱导2h左右,在1630cm-1处检测到明显的吸收峰,该位置的吸收主要来自于聚集体的贡献。进一步对酰胺Ⅰ带区域进行傅里叶去卷积处理,结合高斯曲线拟合,发现在Sc样品中随着诱导时间的增加,聚集体含量逐渐增加,这与SDS-PAGE结果一致。此外,伴随着聚集体的形成,α-螺旋的含量逐渐增加;而β-折叠和无规卷曲的含量却逐渐减少。在S样品中观察到无规卷曲的含量随诱导时间的增加逐渐提高,而β-转角及(1629±1)cm-1处对应的β-折叠的含量却逐渐减少。上述现象表明:C-末端酸性蛋白的引入导致表达过程中β-折叠和无规卷曲逐渐向聚集体和α-螺旋转化。更多还原

【Abstract】 Fourier transform infrared(FTIR)microspectroscopy was used to investigate the effects of C-terminal acidic protein on the secondary structure of wheat α-thionin in the absence of signal peptide during the prokaryotic expression process.SDS-PAGE analysis revealed that the presence of acidic protein gave rise to the formation of inclusion body,however,the absence of acidic protein greatly enhanced the solubility of the heterogenous protein expressed in E.coli BL21(DE3)with the induction of 1 mmol·L-1 IPTG at 37 ℃.Difference spectra in amide I region were obtained by subtraction between the spectra of intact cells containing S and Sc,which corresponds to the absence and presence of C-terminal acidic proteins,respectively.The second derivative of the difference spectra measured 2 h after induction showed one principal component at ～1 630 cm-1,while no significant peak appeared at the same peak position when the spectra before induction were compared.Combined with SDS-PAGE of recombinant protein,the authors presumed that the peak absorption at ～1 630 cm-1 is most likey to be assigned to protein aggregate within inclusion body.Gaussian curve-fitting was done on the Fourier self-deconvolution spectra within amide I region of intact cells containing S and Sc.The experimental data revealed that the relative content of aggregate absorption at(1 629±1)cm-1 gradually increased with induction time,which is consistent with the results of SDS-PAGE.Simutaneously,the formation of aggregate gave rise to the increase of α-helix,as well as the decrease of β-turn and random coil in the case of Sc.It was not the case for S,however,where random coil experienced the increase in the relative average fractions,while β-turn and β-sheet at(1 629±1)cm-1 behaved in different ways.The above mentioned phenomenon indicated that β-sheet and random coil are most likely to transform into aggregate and α-helix with the introduction of C-terminal acidic protein.更多还原