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摘要：安琪酵母（Angel Yeast）是我们生活中常见的一种生物制品。选择酿酒酵母（Saccharomyces cerevisiae）品种，但里面所含的酵母菌可能太杂，所以实验前我们需要先对菌种进行活化并通过划线培养挑选出活性较高的单一菌落进行扩大培养。培养期间，在不同时间段取样运用分光光度计测其OD600。记录绘制生长曲线。酵母Tsa2p蛋白是一种应力诱导胞质硫氧还蛋白过氧化物酶，Tsa2p蛋白存在于酵母菌细胞质中，要提取tsa2p蛋白需先对酵母进行破壁处理。取OD600 约1.0的菌液，加抽提裂解液并加入玻璃珠涡流处理进行破壁，破壁后获得的蛋白是酵母菌的多种蛋白的混合液。取上述蛋白液加入硫酸铵进行盐析，采用盐析法对蛋白进行初步的提纯，初步提纯的蛋白质除了含有Tsa2p蛋白还可能含有其他蛋白质杂质，再运用凝胶层析法对蛋白质进行精提纯。将层析法所得的洗脱液用SDS-PAGE电泳法检测Tsa2p蛋白的提取效果。33762
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毕业论文关键词：酵母培养；Tsa2p蛋白；凝胶层析；SDS-PAGE电泳
The Extraction of Yeast Tsa2p Protein
Abstract: The Angel Yeast (Angel Yeast) is a common kind of biological products in our lives. Brewers yeast (Saccharomyces cerevisiae) varieties were selected as the materials. Because miscellaneous exist in the yeast. We need firstly to activate strains before experiment and screen more active single colony by line to culture it. During the period of yeast strain proliferation, The value of optical density (OD) of the cultures were measured at 600 nm using spectrophotometer. The growth curve were draw.Yeast Stress-induced cytoplasmic Tsa2p protein is a kind of thioredoxin peroxidase, and Tsa2p protein exists in cytoplasm of yeast: To extract Tsa2p proteins need to break the wall of the yeast. Collect yeast liquid at OD600 1.0, then add extraction pyrolysis liquid and glass beads; eddy the mixed yeast to obtain the broken cell contents, Crude proteins were salted out with ammonium sulfate. Preliminary purified proteins in addition to Tsa2p protein may also contain other impurities, then using the gel chromatography the preliminarily extrated protein were further purified. The purified Tsa2p protein was detected using SDS-PAGE.
Key words: Yeast culture; Tsa2p protein; Gel chromatography; SDS-PAGE electrophoresis.
目 录
摘 要 4
引言 5
1.材料和方法 6
1.1 原料 6
1.2缓冲液与培养基 6
1.3 主要试剂材料 8
1.4主要仪器 8
2 试验方法步骤 8
2.1酵母菌的选筛 9
2.1.1YPD培养基的配制 9
2.1.2酵母活化 9
2.1.3酵母菌的初筛 9
2.1.4酵母菌的复筛 9
2.1.5酵母菌的扩大培养 9
2.2蛋白质的提取 10
2.3 Tsa2p蛋白的纯化 10
2.3.1盐析 10
2.3.2凝胶层析提取酵母中的Tsa2p蛋白 10
2.4 SDS-PAGE电泳法检测Tsa2p蛋白 11
3.结果与分析 11
3.1酵母菌分离结果 11
3.2酵母菌纯化结果 12
3.3酵母菌生长曲线 12
3.4蛋白质的提取 13
4. 结论及展望 14
参考文献 15
致谢 16
酵母Tsa2p蛋白的提取
引言
酵母Tsa2p蛋白是一种应力诱导胞质硫氧还蛋白过氧化物酶。Tsa2p蛋白的的相对分子质量为21,615Da，等电点为7.14。硫氧还蛋白过氧化物酶蛋白属于过氧化物酶家族，是一类新近发现的过氧化物酶，广泛分布于真核和原核生物中[1]。与其它的过氧化物酶有所不同的是它缺少催化反应通常所需的含金属离子的辅基，而具有1-2个保守的Cys 残基，可以替代金属离子辅基的功能，参与各种过氧化物的清除反应，从而提生物的抗逆性[2]。硫氧还蛋白过氧化物酶不依赖于敏感性的辅因子，可以清除广泛的过氧化物。硫氧还蛋白过氧化物酶的催化活性和氨基酸序列与其它抗氧化酶类完全不同，在氧化应激反应中起着至关重要的作用。