应用优化的双向电泳技术建立纤维堆囊菌So0157-2蛋白质组数据库(英文)

Establishment of Sorangium cellulosum So0157-2 Proteome Database Using Optimized Two-dimensional Electrophoresis Protocol

堆囊菌丰富的次级代谢产物是新药的重要来源,而蛋白质组学分析是研究代谢调控的有效方法.然而堆囊菌含有大量的胞外多糖以及黏液,干扰了蛋白质组学分析中蛋白质的溶解度、分辨率及重现性.为了高通量地筛选Sorangium cellulosumSo0157-2表达的特异性蛋白,实验优化了S.cellulosum So0157-2双向电泳方法.首先,S.cellulosum So0157-2蛋白在裂解液中有更好的溶解度.pH 3～10非线性胶条和1 mg的蛋白上样量适用于第一向等电聚焦,分别提高了蛋白质点的分辨率和低丰度蛋白质的表达.15%SDS-PAGE改善了S.cellulosum So0157-2蛋白分离的分辨率和重现性.最终,通过优化的双向电泳方法获得了S.cellulosum So0157-2在M26培养基中培养3天的全蛋白质表达谱,并检测到552个蛋白质点.进而对表达蛋白通过MALDI-TOF-MS进行质谱鉴定,其中474个蛋白质得到鉴定,鉴定率85.9%.得到鉴定的蛋白质包括细胞结构和功能组分,以及细胞代谢合成酶类,其中8个蛋白质与糖类的转化和代谢相关,这有助于糖基化埃博霉素A的深入研究.该优化方法为进一步建立纤维堆囊菌So0157-2在各种培养条件下的蛋白质组表达数据库打下基础.

The rich classes of secondary metabolites from the genus Sorangium have been an important source of new drugs. The proteome analysis is an effective method to study the regulation of metabolism. However, the genus Sorangium contains a large amount of exopolysaccharides or slime that interferes with protein solubility, resolution, and repeatability in proteome analysis. To perform high-throughput screening of the specific proteins expressed by Sorangium cellulosum So0157-2, we optimized the two-dimensional electrophoresis （2-DE） protocol. Firstly, the proteins have better solubility in lysis buffer. The pH 3~10 NL strip is appropriate for the first-dimensional isoelectric focusing, improving the resolution of protein spots. 1 mg of protein was used in the isoelectric focusing, improving the expression of low-accumulation proteins. 15% SDS-PAGE improved the resolution and repeatability for separation of these proteins. Based on the optimal 2-DE protocol, the protein patterns ofS. cellulo.sum So0157-2 cultured in M26 medium for three days were acquired, and 552 protein spots were detected. Further, the expressed proteins （85.9%） were identified by MALDI-TOF-MS. The identified proteins included components of cell structure and function, and cell metabolic enzymes. Worthy to be mentioned, 8 proteins were related to the transformation and metabolism of carbohydrate, which were contributed to the in-depth study of epothiloneoside A. This optimal protocol laid the foundation for the further construction of proteome expression database of S. cellulosurn So0157-2 in various industrial culture conditions.