乳杆菌胞外蛋白提取鉴定与双向电泳图谱建立

Extraction and Identification of Extracellular Proteins from Lactobacilli and a Reference Extracellular Proteomic Map of Lactobacillus paracasei L14

摘要:

采用三氯乙酸（TCA）-丙酮沉淀法提取乳杆菌胞外蛋白并进行了优化，通过SDS-PAGE分析了该方法用于7株乳杆菌胞外蛋白的提取效果，同时对乳杆菌不同生长期胞外蛋白的分泌特性进行了研究，采用MALDI-TOF/TOF-MS鉴定了胞外蛋白，最后通过双向电泳(2-DE)分析了L. paracasei L14的胞外蛋白组。结果表明：采用终质量分数为6%的TCA提取乳杆菌胞外蛋白效果最好，并且具有通用性；从乳杆菌稳定期提取出种类较多的胞外蛋白，成功鉴定出L. paracasei SW2胞外蛋白中3个蛋白质；使用半合成培养基培养乳杆菌，可以提取出符合2-DE样品纯度要求的胞外蛋白，从L. paracasei L14胞外蛋白组2-DE图谱上检测到(130±10)个蛋白质点，约占L. paracasei预测分泌蛋白组的46%。

Abstract:

Lactobacillus extracellular proteins are key molecular factors in promoting the probiotichost crosstalk and signal transduction. Their extractions and identifications could contribute to unravelling the probiotic molecular mechanisms such as adhesion to intestinal surfaces, modulating immune response and enhancing antagonism towards pathogens. A protocol based on trichloroacetic acid (TCA)acetone precipitation of proteins was presented and optimized. The method was tested on seven Lactobacillus strains after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The extracellular proteins secretion capacity of L. paracasei SW2 in different growth phases was studied and some protein bands were selected for identification by tandem MS. The extracellular proteome of L. paracasei L14 were analyzed by 2-D electrophoresis (2-DE). The results showed that the final concentration of 6% TCA was the most suitable for extracting extracellular proteins from lactobacilli. More extracellular proteins could be extracted in the stationary phase than midexponential growth phase. Three of extracellular proteins extracted from L. paracasei SW2 were successfully identified. The extracellular samples extracted from semidefined medium (SDM) could be analyzed by 2-DE. 130±10 spots, corresponding to about 46% coverage of the predicted secretome of L. paracasei, were detected in the 2-DE map of L. paracasei L14 extracellular proteome. In conclusion, this study preliminarily established the extraction method and 2-DE analysis system of extracellular proteins from lactobacilli and paved the way for a more comprehensive insight into relevant research.