酿酒酵母总蛋白的提取及其耐核苷类药物表达特性分析

发布时间：2018-06-30 00:38

本文选题：酿酒酵母 + 细胞破碎　；参考：《实验室研究与探索》2016年08期

【摘要】：建立了一种简便适用于从真核细胞酿酒酵母菌中提取总蛋白的方法,应用该方法探索了两株对核苷类药物5-FC高耐受菌株的蛋白表达特性。实验中以细胞破碎率和总蛋白浓度为主要检测指标,对比分析了6种酵母细胞破碎方法,即液氮研磨、超声波破碎、酸洗玻璃珠法、酶法、反复冻融法、TNBIO高压细胞破碎,通过比色法测定了总蛋白的含量,发现改进后的酸洗玻璃珠法的细胞破碎率为81.25%,总蛋白含量152.67 mg/m L,效果明显优于其他几种方法.实验表明,在裂解液中添加适量的PMSF等蛋白酶抑制剂,可以有效防止蛋白的降解。经过SDSPAGE对核苷类药物高耐药性菌株酵母总蛋白表达特性分析,酸洗玻璃珠法提取的蛋白含量高,所含杂质少,条带比较多且清晰,其中相对分子质量约为21 kDa和43kDa的蛋白有明显的表达差异,可能与菌株细胞的耐药性相关。
[Abstract]:A simple and convenient method for extracting total protein from eukaryotic Saccharomyces cerevisiae was established. The protein expression characteristics of two strains with high tolerance to nucleoside drugs 5-FC were investigated. In the experiment, the cell breakage rate and the total protein concentration were used as the main indexes to compare and analyze six kinds of yeast cell breaking methods, namely, liquid nitrogen grinding, ultrasonic crushing, acid washing glass bead method, enzymatic method, repeated freezing thawing method and TNBIO high pressure cell fragmentation. The content of total protein was determined by colorimetry. It was found that the cell fragmentation rate of the improved acid pickling glass bead method was 81.25 and the total protein content was 152.67 mg/m / L. the effect was obviously superior to that of other methods. The results showed that protease inhibitors such as PMSF could effectively prevent protein degradation. After SDSPAGE analysis of the expression characteristics of total protein in yeast with high resistance to nucleoside drugs, it was found that the protein extracted by acid washing glass bead method had a high content, less impurities and more bands. The protein with relative molecular weight of about 21 kDa and 43 kDa was significantly different, which may be related to the resistance of strain cells.
【作者单位】：东华大学化学化工与生物工程学院;
【基金】：中央高校基本科研业务费专项资金项目(2232014A3-03) 上海市自然科学基金项目(15ZR1400200)
【分类号】：R915

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