小麦叶片类囊体膜蛋白复合体的分离和分析技术研究

Technological Study on Separation and Analysis of Thylakoid Membrane Protein Complexes from Wheat Leaves

膜蛋白复合体在生命活动中执行着重要的生物化学功能,具有重要的研究意义,但由于其低表达和高度疏水的特点,完整状态和低丰度的膜蛋白复合体的分离分析仍是一大挑战。首先,采用蓝绿温和胶非变性电泳作为第一维分离技术,大量分离制备叶绿体类囊体膜蛋白复合物;其次,采用电洗脱方法差异富集第一维电泳分离到的各种蛋白复合体;最后,采用SDS-PAGE作为第二维分离技术,分析差异富集的不同种蛋白复合体的亚基构成。研究结果表明,利用这种分离分析模式,能有效地富集膜蛋白复合体尤其是低丰度膜蛋白复合体,明显提高第二维分离分析的分辨率和蛋白覆盖率。

Membrane protein complexes perform important biochemical function in cellular process,but the analysis of membrane protein complexes is a significant challenge due to their hydrophobic properties and relatively low abundance.In this study,blue-native polyacrylamide gel electrophoresis（BN-PAGE）was used to separate and prepare a large number of chloroplast thylakoid membrane protein complexes in the first dimension.Then,electroelution was performed to differentially enrich various native protein complexes separated in the first dimension.Electroeluted protein complexes were analyzed to determine the subunit composition with sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） in the second dimension.The application of the BN/SDS-PAGE strategy to the separation and analysis of membrane protein complexes,especially in relative low-abundance,demonstrated that a high resolution and protein coverage was achieved by enrichment of low-abundant membrane protein and separation of complexes subunits in the second-dimensional SDS/PAGE.