CBM2蛋白的高密度自诱导

doi:10.11924/j.issn.1000-6850.casb15020102

中国农学通报 ›› 2015, Vol. 31 ›› Issue (18): 140-145.doi: 10.11924/j.issn.1000-6850.casb15020102

所属专题：生物技术

CBM2蛋白的高密度自诱导

马闪闪,韩来闯,刘亚娟,刘猛,刘亮伟

(河南农业大学生命科学学院,郑州 450002）

收稿日期:2015-02-26 修回日期:2015-06-11 接受日期:2015-05-14 出版日期:2015-07-27 发布日期:2015-07-27
通讯作者: 刘亮伟
基金资助:
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)。

High Density Self-induction of CBM2

Ma Shanshan, Han Laichuang, Liu Yajuan, Liu Meng, Liu Liangwei

(College of Life Science, Henan Agricultural University, Zhengzhou 450002)

Received:2015-02-26 Revised:2015-06-11 Accepted:2015-05-14 Online:2015-07-27 Published:2015-07-27

摘要/Abstract

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200 μL ZYM-5052菌体细胞含有82.3 μg CBM2，占细胞总蛋白55.3%；等量IPTG LB菌体细胞含有16.6 μg 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。

关键词: 生防细菌, 生防细菌, 根际, 微生态

Abstract: 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-lactose produced 82.3 μg CBM2, consisting 55.3% of the total protein. 200 μL cells incubated with LB-IPTG produced 16.6 μg 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-lactose. 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-lactose 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.

马闪闪,韩来闯,刘亚娟,刘猛,刘亮伟. CBM2蛋白的高密度自诱导[J]. 中国农学通报, 2015, 31(18): 140-145.

Ma Shanshan,Han Laichuang,Liu Yajuan,Liu Meng and Liu Liangwei. High Density Self-induction of CBM2[J]. Chinese Agricultural Science Bulletin, 2015, 31(18): 140-145.

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CBM2蛋白的高密度自诱导

High Density Self-induction of CBM2

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