| 摘要: |
| 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。 |
| 关键词: 钝顶节旋藻, 蛋白质, 制备方法, 双向电泳, 螺旋手性 |
| DOI：10.11693/hyhz201301021021 |
| 分类号: |
| 基金项目:国家自然科学基金项目, 30771669 号, 10975118 号; 国家公益性行业(农业)科研专项, 201107003 号; 浙江省院士基金, J20080388号, J20110445 号。 |
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| AN IMPROVED METHOD OF 2-DE ANALYSIS PROTEIN PREPARATION AND USING FOR INVESTIGATION OF HELICAL-HANDED DIFFERENTIAL PROTEINS IN ARTHROSPIRA PLATENSIS |
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WANG Jing-Mei, WANG Zhi-Ping, YU Jin-Xin, LIU Xin-Ying, SHAO Bin, LAN Jin-Jin, MA Li-Fang, CHEN Zi-Yuan
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Institute of Nuclear Agriculture Sciences, Zhejiang University, Key Laboratory of Nuclear Agricultural Sciences, Ministry of Agriculture of China
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| Abstract: |
| 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-HCl 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 toeffective 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, environmental 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. |
| Key words: Arthrospira platensis, Protein, Preparation method, Two-dimensional gel electrophoresis, Helical chirality |
