CBM2蛋白的高密度自诱导  

High Density Self-induction of CBM2

作　　者：马闪闪[1] 韩来闯 刘亚娟[1] 刘猛[1] 刘亮伟[1] 

机构地区：[1]河南农业大学生命科学学院,郑州450002

出　　处：《中国农学通报》2015年第18期140-145,共6页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金"基于结构分析的嗜热CBM拆分-易位-组合融合对木聚糖酶稳定性-活性影响"(31371831);"基于pH相关氨基酸定位分析的双功能木聚糖酶耐酸性突变研究"(30972123)

摘　　要：为提高外源蛋白表达量,以pET20b载体、BL21(DE3)E.coli宿主细胞,比较了乳糖和IPTG对CBM2(碳水化合物结合结构域)蛋白表达量的诱导效果。ZYM-5052自诱导培养基菌体OD600是IPTG LB培养基菌体的4.4倍。SDS-PAGE显示200μL ZYM-5052菌体细胞含有82.3μg CBM2,占细胞总蛋白55.3%;等量IPTG LB菌体细胞含有16.6μg CBM2,占细胞总蛋白25.7%。取不同培养时间等量等同OD600的ZYM-5052菌体进行SDS-PAGE,结果显示单位细胞中蛋白量随着培养时间而增加。所以高密度自诱导不仅提高了细胞生长密度、而且提高了单位细胞中蛋白表达量。自诱导方案用于表达葡聚糖酶,与IPTG LB菌体相比,自诱导菌体OD600是其2.3倍,酶活性是其4.5倍。该研究为提高外源蛋白表达量提供了较为简单、普适的高密度自诱导方案。To improve foreign protein production in E. coli, effects of lactose and IPTG were compared on production of carbohydrate-binding module （CBM2） by employing pET20b as expression vector and BL21 （DE3） E.coli as host cell. OD600 value of cells incubated in ZYM-5052 media with lactose was 4.4-fold of those incubated in LB media with IPTG. After SDS-PAGE analysis, 200 μL cells incubated with ZYM-5052-1actose produced 82.3 μg CBM2, consisting 55.3% of the total protein. 200 μL cells incubated with LB-IPTG produced 16.6 pg CBM2, consisting 25.7% of the total protein. SDS-PAGE of cells with equivalent OD600 value showed that CBM2 increased along with time in each cell incubated in ZYM-5052-1actose. Thereby, high- density self-induction method increased both cell density and protein production in each ccell. High-density self-induction method was also used to express glucanase, and the results showed that OD600 value and glucanase activity of cells incubated with ZYM-5052-1actose were 2.3-fold and 4.5-fold of those incubated with LB-IPTG. The study provided a relative simple and more general high-density self-induction method to increase foreign protein production.

关 键 词：CBM2 高密度 自诱导 

分 类 号：Q556[生物学—生物化学]