东方伊萨酵母降解染料蛋白质组的双向电泳条件优化

Optimization of Two-dimensional Electrophoresis Conditions for Proteomics of Issatchenkia orientalis in Degrading Dyes

利用双向凝胶电泳技术分离东方伊萨酵母Issatchenkia orientalis降解染料活性艳红K-2BP前后细胞内总蛋白质,并比较不同样品处理方法、不同上样量以及不同染色方法等对电泳结果的影响,进而获得较优的分离染料降解相关蛋白的双向凝胶电泳条件.在3种样品处理方法中,单纯超声波破碎,超声波-TCA/丙酮沉淀,超声波-硫酸铵沉淀获得的最终总蛋白浓度分别为3.4、1.8和5.6 mg/mL.电泳结果表明,采用超声波-硫酸铵沉淀提取蛋白样品,用固相pH 4～7梯度胶条进行等点聚焦,上样量150μg,电泳结束后硝酸银染色,最终获得了分辨率高和重复性好的双向电泳图谱.经过初步图像分析,检测到730±30个蛋白点.该研究为利用蛋白质组学技术筛选酵母菌降解染料过程中存在的功能酶蛋白提供了技术支持.

Total protein of the yeast Issatchenkia orientalis was extracted and separated by two-dimensional electrophoresis（2-DE） before and after the dye Reactive Brilliant Red K-2BP was degraded by this yeast,respectively.Different protein extraction methods,different volumes of sample loaded and different staining techniques were tested and compared for the 2-DE.Among three different protein extraction methods,the final protein concentrations of 3.4 mg/mL,1.8 mg/mL and 5.6 mg/mL were obtained by single ultrasonication,ultrasonication-TCA/acetone,and ultrasonication-ammonium sulfate precipitation,respectively.The best electrophoresis pattern could be gotten by loading 150 μg protein samples from the method of ultrasonication-ammonium sulfate precipitation,using IPG strips of pH 4-7 for the first dimensional electrophoresis and staining with silver nitrate.This electrophoresis pattern had high resolution and good repetition.It was detected to have 730 ± 30 protein points by preliminary image analysis.This research results provided a technical support for screening dye-degrading enzymes from the yeast of I.orientalis.