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傅里葉顯微紅外研究C-末端酸性蛋白對α-硫素原核表達過程的影響
摘要：采用傅里葉變換顯微紅外手段研究了在信號肽缺失的情況下,C-末端酸性蛋白的存在對小麥α-硫素原核表達過程中蛋白質二級結構的影響.SDS-PAGE表明帶有酸性蛋白(Sc樣品)的重組質粒在大腸桿菌BL21(DE3)中以包涵體形式高效表達;而酸性蛋白缺失(S樣品)可以極大提高外源蛋白的可溶性.通過對含有S及Sc的全細胞在誘導前及誘導后2 h的酰胺Ⅰ帶區(qū)域圖譜求籌譜及二階導數(shù)譜,發(fā)現(xiàn)誘導前在1 630cm~1處吸收較弱,而在誘導2 h左右,在1 630 cm~(-1)處檢測到明顯的'吸收峰,該位置的吸收主要來自于聚集體的貢獻.進一步對酰胺Ⅰ帶區(qū)域進行傅里葉去卷積處理,結合高斯曲線擬合,發(fā)現(xiàn)在Sc樣品中隨著誘導時間的增加,聚集體含量逐漸增加,這與SDS-PAGE結果一致.此外,伴隨著聚集體的形成,α-螺旋的含量逐漸增加;而β-折疊和無規(guī)卷曲的含量卻逐漸減少.在S樣品中觀察到無規(guī)卷曲的含量隨誘導時間的增加逐漸提高,而β-轉角及(1 629±1)cm~(-1)處對應的β-折疊的含量卻逐漸減少.上述現(xiàn)象表明:C-末端酸性蛋白的引入導致表達過程中β-折疊和無規(guī)卷曲逐漸向聚集體和α-螺旋轉化.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 containing S and Sc, which corresponds to the absence and presence of C-terminal acidic proteins, respectively. The second de-rivative of the difference spectra measured 2 h after induction showed one principal component at ～1 630 cm~(-1) , while no signifi-cant peak appeared at the same peak position when the spectra before induction were compared. Combined with SD～PAGE of recombinant protein, the authors presumed that the peak absorption at ～1 630 cm~(-1) is most likey to be assigned to protein ag-gregate 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 a-helix, as well as the decrease of fl-turn and random coil in the ease 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 fl-sheet and random coil are most likely to transform into aggregate and α-helix with the introduction of C-terminal acidic protein. 作者： 劉艷[1]馮娟[1]陶棟梁[2]翁詩甫[2]任正隆[3] Author： LIU Yan[1] FENG Juan[1] TAO Dong-liang[2] WENG Shi-fu[2] REN Zheng-long[3] 作者單位： 電子科技大學生命科學與技術學院,四川成都,610054北京大學化學與分子工程學院,北京,100871電子科技大學生命科學與技術學院,四川成都,610054;四川農(nóng)業(yè)大學植物遺傳育種省級蓖點實驗室,四川雅安,625014 期 刊： 光譜學與光譜分析 ISTICEISCIPKU Journal： SPECTROSCOPY AND SPECTRAL ANALYSIS 年，卷(期)： 2009, 29(12) 分類號： Q657.3 關鍵詞： 顯微紅外 C-末端酸性蛋白 α-硫素 原核表達 二級結構 Keywords： FTIR microspectroscopy C-terminal acidic protein α-thionin Prokaryotic expressio Secondary structure 機標分類號： R73 O6 機標關鍵詞： 傅里葉 顯微紅外 酸性蛋白 硫素 原核表達 過程 Prokaryotic Expression Acidic Protein Study acidic protein SDS-PAGE inclusion body aggregate 誘導時間 無規(guī)卷曲 聚集體 prokaryotic expression spectra 含量 secondary structure 基金項目： 國家自然科學基金重點項目，電子科技大學青年基金重點項目【傅里葉顯微紅外研究C-末端酸性蛋白對α-硫素原核表達過程的影響】相關文章：
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