Study On Extracellular Protein Of Saccharomyces Cerevisiae

Secretory protein refers to the secretion of cell or tissue to the extracellular protein, compatriots protein involved in cell proliferation, differentiation and cell signal transduction and other important life processes. Therefore, this topic explores a new kind of extracellular protein extraction and analysis method, based on Saccharomyces cerevisiae extracellular proteomics, to reveal the Saccharomyces cerevisiae extracellular proteomics changes under the condition of continuous training aging.This article selected two-dimensional electrophoresis technology as the main techniques of proteomics analysis. The key to apply this technology in extracellular proteomics was the preparation of the protein sample. Saccraomyces cerevsiae FFC2144 was cultured in nitrogen base without protein medium. The total proteins of yeast were extracted by ammonium sulfate precipitation, ultrafiltration and lyophilization-phenol extraction. Extraction rate were calculated and the proteins were separated by two-dimensional electrophoresis. The proteins isolated were confirmed by MALDI-TOF-MS. The extraction rate of lyophilization-phenol method was 73.67% and 114 protein spots were obtained after two-dimensional(2-D) electrophoresis profiling to identify. The method of Lyophilized-phenol extraction in the possession of high extraction rate, sufficient protein spots identification and clear electrophoretogram than other methods more suitable for two-dimensional(2-D) electrophoresis analysis of yeast extracellular proteins which is an ideal method of secretory proteomics research. Second ultrafiltration method, ammonium sulphate precipitation because have too much salt is bad for two-dimensional electrophoresis.In order to expound the secretory protein changes during yeast aging. The yeast were cultivated in saline while determined the extracellular protein content, activities of antioxidant enzymes and protease. We evaluated the effect of long term culture on the senescence of these cells, total extracellular proteins were extracted and separated by two-dimensional electrophoresis, compared with normal(YNB) culture of extracellular protein. Then the different points between them were identified by MALDI-TOF-MS. Under the condition of continuous aging the intracellular antioxidant enzyme activity was reduced, while the activity of protease and extracellular protein went up at the same time. The MALDI-TOF-MS shows that extracellular protein contains some intracellular protein fragments. It may be related to the increasing activities of protease in cells, intracellular "useless proteins" were hydrolyzed into pieces to extracellular environment.