傅里葉顯微紅外研究C-末端酸性蛋白對(duì)α-硫素原核表達(dá)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(hào)肽缺失的情況下,C-末端酸性蛋白的存在對(duì)小麥α-硫素原核表達(dá)過程中蛋白質(zhì)二級(jí)結(jié)構(gòu)的影響.SDS-PAGE表明帶有酸性蛋白(Sc樣品)的重組質(zhì)粒在大腸桿菌BL21(DE3)中以包涵體形式高效表達(dá);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(duì)含有S及Sc的全細(xì)胞在誘導(dǎo)前及誘導(dǎo)后2 h的酰胺Ⅰ帶區(qū)域圖譜求籌譜及二階導(dǎo)數(shù)譜,發(fā)現(xiàn)誘導(dǎo)前在1 630cm~1處吸收較弱,而在誘導(dǎo)2 h左右,在1 630 cm~(-1)處檢測(cè)到明顯的吸收峰,該位置的吸收主要來自于聚集體的貢獻(xiàn).進(jìn)一步對(duì)酰胺Ⅰ帶區(qū)域進(jìn)行傅里葉去卷積處理,結(jié)合高斯曲線擬合,發(fā)現(xiàn)在Sc樣品中隨著誘導(dǎo)時(shí)間的增加,聚集體含量逐漸增加,這與SDS-PAGE結(jié)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(dǎo)時(shí)間的增加逐漸提高,而β-轉(zhuǎn)角及(1 629±1)cm~(-1)處對(duì)應(yīng)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(dǎo)致表達(dá)過程中β-折疊和無規(guī)卷曲逐漸向聚集體和α-螺旋轉(zhuǎn)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(shī)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(xué)生命科學(xué)與技術(shù)學(xué)院,四川成都,610054北京大學(xué)化學(xué)與分子工程學(xué)院,北京,100871電子科技大學(xué)生命科學(xué)與技術(shù)學(xué)院,四川成都,610054;四川農(nóng)業(yè)大學(xué)植物遺傳育種省級(jí)蓖點(diǎn)實(shí)驗(yàn)室,四川雅安,625014 期 刊： 光譜學(xué)與光譜分析   ISTICEISCIPKU Journal： SPECTROSCOPY AND SPECTRAL ANALYSIS 年，卷(期)： 2009, 29(12) 分類號(hào)： Q657.3 關(guān)鍵詞： 顯微紅外    C-末端酸性蛋白    α-硫素    原核表達(dá)    二級(jí)結(jié)構(gòu)    Keywords： FTIR microspectroscopy    C-terminal acidic protein    α-thionin    Prokaryotic expressio    Secondary structure    機(jī)標(biāo)分類號(hào)： R73 O6 機(jī)標(biāo)關(guān)鍵詞： 傅里葉    顯微紅外    酸性蛋白    硫素    原核表達(dá)    過程    Prokaryotic Expression    Acidic Protein    Study    acidic protein    SDS-PAGE    inclusion body    aggregate    誘導(dǎo)時(shí)間    無規(guī)卷曲    聚集體    prokaryotic expression    spectra    含量    secondary structure 基金項(xiàng)目： 國(guó)家自然科學(xué)基金重點(diǎn)項(xiàng)目，電子科技大學(xué)青年基金重點(diǎn)項(xiàng)目

【傅里葉顯微紅外研究C-末端酸性蛋白對(duì)α-硫素原核表達(dá)過程的影響】相關(guān)文章：

TRAIL蛋白原核表達(dá)載體的構(gòu)建及表達(dá)研究05-02

原核生物中硒蛋白表達(dá)的研究進(jìn)展05-02

重組SARS冠狀病毒S蛋白的原核表達(dá)研究05-02

人朊蛋白基因的克隆與原核表達(dá)04-27

綿羊重組朊蛋白的原核表達(dá)與結(jié)構(gòu)分析05-02

GST-talin1融合蛋白的原核表達(dá)及純化04-27

人cubilin蛋白功能片段原核表達(dá)系統(tǒng)的建立04-28

C-反應(yīng)蛋白的研究進(jìn)展04-26

酸弗林蛋白酶酸性氨基酸簇分選蛋白-1的原核表達(dá)、純化及多克隆抗體制備04-27

新城疫病毒F和HN蛋白的原核表達(dá)04-27