High-level autoenhanced expression of a single-copy gene in Escherichia coli:: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements
High-level autoenhanced expression of a single-copy gene in Escherichia coli:: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements
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DOI：
10.1016/s0378-1119(00)00526-6
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发表时间：
2001-01-10
期刊：
影响因子：
3.5
通讯作者：
Dreyfus, M
中科院分区：
文献类型：
--
作者：
Marchand, I;Nicholson, AW;Dreyfus, M
噬菌体T7早期基因0.7帮助噬菌体在次优条件下生长(辅助功能)。C端1/3的编码蛋白参与宿主转录关闭，N端2/3具有广泛特异性的蛋白激酶(‘PK’)活性。然而，这一活动与助手功能的关系尚不清楚。在这里，编码PK的0.7个截短基因与IPTG可诱导的T7晚期启动子和T7晚期基因的翻译起始区融合，并插入到表达T7 RNA聚合酶的大肠杆菌的染色体上。诱导后，总蛋白质合成保持不变，但40%以上用于PK合成，对于单拷贝基因的表达来说，这是一个惊人的数字。取消PK活性的突变使这种表达减少了3倍。因此，当后者受T7晚期遗传元件控制时，PK活性会刺激PK的表达。进一步的实验表明，刺激发生在转录和转录后水平。因此，辅助功能可能对应于PK介导的对晚期表达的刺激，其机制进行了讨论。此外，还考虑了利用PK活性改进大肠杆菌表达系统的可能性。(C)2001 Elsevier Science B.V.保留所有权利。
Bacteriophage T7 early gene 0.7 assists phage growth under suboptimal conditions ('helper' function). Whereas the C-terminal one-third of the encoded protein participates in host transcription shutoff, the N-terminal two-thirds harbours a protein kinase ('PK') activity with broad specificity. However, how this activity relates to helper function is unclear. Here, a truncated gene 0.7 encoding PK was fused to an IPTG-inducible T7 late promoter and to a translation initiation region from a T7 late gene, and inserted into the chromosome of an E. coli strain expressing T7 RNA polymerase. After induction, total protein synthesis remains unchanged but with over 40% devoted to PK synthesis, an amazing figure for the expression of a single-copy gene. Mutations abolishing PK activity reduce this expression by 3-fold. Thus, PK activity stimulates PK expression when the latter is controlled by T7 late genetic elements. Further experiments show that stimulation occurs at both transcriptional and post-transcriptional levels. The helper function may therefore correspond to a PK-mediated stimulation of late expression, the mechanism of which is discussed. The possibility of exploiting the PK activity fur improving E. coli expression systems is also considered. (C) 2001 Elsevier Science B.V. All rights reserved.