钝顶节旋藻(Arthrospira platensis)2-DE 分析蛋白制备方法改进及试用于螺旋手性差异蛋白研究

AN IMPROVED METHOD OF 2-DE ANALYSIS PROTEIN PREPARATION AND USING FOR INVESTIGATION OF HELICAL-HANDED DIFFERENTIAL PROTEINS IN ARTHROSPIRA PLATENSIS

采用冻融破壁和蛋白质分步提取等技术,对钝顶节旋藻2-DE分析蛋白制备方法作了改进,并试用于螺旋手性差异表达蛋白研究,结果表明:(1)钝顶节旋藻细胞反复冻融5次完全破碎,用Tris-HCl提取液提取3次可提得近87%的水溶性蛋白,再用SDS(十二烷基磺酸钠)提取液可从沉淀中提得水不溶性蛋白;(2)将上述水溶性和水不溶性蛋白分别用TCA/丙酮法纯化后作双向电泳(2-DE)分析,可辨蛋白点分别达500和760多个,而两者间的匹配率仅7%,说明分离度好且质量高;(3)利用此法获得了13个与钝顶节旋藻螺旋手性相关的候选蛋白,其中5个为首次发现,候选蛋白功能涉及光合作用、能量代谢、物质外排和环境适应等;(4)本文所建的"冻融破壁分步提纯法"与现有节旋藻2-DE蛋白制备方法相比,具有操作简便、仪器设备简单、成本低廉及信息量更丰富等优点。

Based on the techniques of cell wall breaking by freeze-thaw and proteins extracting step-by-step, protein preparation method for 2-DE analysis of Arthrospira platensis was improved, and then was applied to investigation of helical chirality-related proteins. The results indicated that： （1） After Arthrospira cells were completely ruptured by five cycles of freeze-thaw, about 87% of water-soluble proteins could be extracted by Tris-HC1 extractant for three times, and then water-insoluble proteins were extracted by SDS （twelve sodium dodecyl sulfate） extractant from the precipitate. （2） The above water-soluble and water-insoluble proteins were analyzed by two-dimensional gel electrophoresis （2-DE） after they were purified by the method of TCA/acetone. Owing to effective separation and high quality, the amount of detected protein spots in the water-soluble and water-insoluble protein patterns was more than 500 and 760 respectively, and their matching rate was only 7%. （3） By this method, 13 candidate proteins including five new were identified as related to helical chirality in Arthrospira, and their function were involved in photosynthesis, energy metabolism, exocytosis, envi- ronmental adaptation, etc. （4） Compared to the existing protein preparation methods for 2-DE analysis of Arthrospira, the method of ＂breaking cell wall by freeze-thaw and then extracting step-by-step＂ presented in this paper, has the advantages of simplification in operation and instruments, low cost and more information, and so on.