通过启动子工程和基因剂量策略在大肠杆菌中高效表达中国蝎肽BmK1。
Significant expression of a Chinese scorpion peptide, BmK1, in Escherichia coli through promoter engineering and gene dosage strategy.
作者信息
Wang Jianfeng, Xiong Zhiqiang, Yang Yingying, Zhao Na, Wang Yong
机构信息
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
出版信息
Biotechnol Appl Biochem. 2014 Jul-Aug;61(4):466-73. doi: 10.1002/bab.1194. Epub 2014 Mar 25.
Heterologous expression is an efficient alternative to conventional extraction to produce a specific Buthus martensii Karsch (BmK) peptide. In this work, BmK1 was successfully expressed in Escherichia coli after genetic codon optimization, but BmK1 content was <6% of total cellular protein. To improve BmK1 expression, a trc promoter library with a wide relative strength was constructed, and three promoters, PpJF136 (0.55), PpJF325 (1.29), and PpJF288 (2.31), were selected to control BmK1 expression. A higher BmK1 expression (>13.9% of total protein) was obtained using a stronger promoter, PpJF325 . Furthermore, a maximum BmK1 content (>21.7% of total protein) was obtained by combining promoter PpJF325 and three copies of the BmK1 gene. The yield of the purified BmK1 achieved 196.74 mg L(-1) in E. coli BL21(DE3) pJF431, which was improved 2.09-fold compared with the control. This was the highest reported production of scorpion peptides in E. coli.
异源表达是一种替代传统提取方法的高效手段，用于生产特定的东亚钳蝎（BmK）肽。在本研究中，经过遗传密码子优化后，BmK1在大肠杆菌中成功表达，但BmK1含量占总细胞蛋白的比例小于6%。为提高BmK1的表达水平，构建了一个具有广泛相对强度的trc启动子文库，并筛选出三个启动子PpJF136（0.55）、PpJF325（1.29）和PpJF288（2.31）来调控BmK1的表达。使用更强的启动子PpJF325获得了更高的BmK1表达水平（占总蛋白的比例>13.9%）。此外，通过将启动子PpJF325与三个拷贝的BmK1基因相结合，获得了最高的BmK1含量（占总蛋白的比例>21.7%）。在大肠杆菌BL21(DE3) pJF431中，纯化后的BmK1产量达到196.74 mg L(-1)，与对照相比提高了2.09倍。这是报道的大肠杆菌中蝎子肽的最高产量。
Zotero 插件