[Study on the effect of C-terminal acidic protein on the prokaryotic expression of alpha-thionin by FTIR microspectroscopy].
Fourier transform infrared (FTIR) microspectroscopy was used to investigate the effects of C-terminal acidic protein on the secondary structure of wheat alpha-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 x L(-1) IPTG at 37 degrees C. 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 approximately 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 approximately 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 alpha-helix, as well as the decrease of beta-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 beta-turn and beta-sheet at (1 629 +/- 1) cm(-1) behaved in different ways. The above mentioned phenomenon indicated that beta-sheet and random coil are most likely to transform into aggregate and alpha-helix with the introduction of C-terminal acidic protein.
Liu
Yan
Y
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. liuyan841107@yahoo.com.cn
Feng
Juan
J
Tao
Dong-Liang
DL
Weng
Shi-Fu
SF
Ren
Zheng-Long
ZL
D004926:Escherichia coli; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D054809:Thionins / Q000737:chemistry*
D054809:Thionins