Study on the Effect of C-Terminal Acidic Protein on the Prokaryotic Expression of α-Thionin by FTIR Microspectroscopy
LIU Yan1, FENG Juan1*, TAO Dong-liang3, WENG Shi-fu3, REN Zheng-long1, 2*
1. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China 2. Sichuan Province Key Laboratory of Plant Genetics and Breeding, Sichuan Agricultural University，Yaan 625014，China 3. College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
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.
刘 艳1，冯 娟1*，陶栋梁3，翁诗甫3，任正隆1, 2* . 傅里叶显微红外研究C-末端酸性蛋白对α-硫素原核表达过程的影响[J]. 光谱学与光谱分析, 2009, 29(12): 3267-3270.
LIU Yan1, FENG Juan1*, TAO Dong-liang3, WENG Shi-fu3, REN Zheng-long1, 2* . Study on the Effect of C-Terminal Acidic Protein on the Prokaryotic Expression of α-Thionin by FTIR Microspectroscopy. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2009, 29(12): 3267-3270.
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