Translation enhancement by a Dictyostelium gene sequence in Escherichia coli
Translation enhancement by a Dictyostelium gene sequence in Escherichia coli
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DOI：
10.1007/s00253-019-09746-7
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发表时间：
2019-03
影响因子：
5
通讯作者：
Tomo Kondo;S. Yumura
中科院分区：
文献类型：
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作者：
Tomo Kondo;S. Yumura
大肠杆菌生产异源蛋白质的方法彻底改变了生物技术和生物工业。最终重要的是增加来自细菌的蛋白质产物的量。为此，开发了各种工具，如有效的促进剂。在这里，我们提出了一个通用的分子工具的基础上的现象，称为“翻译增强aDictyosteliumgene序列”（“TED”）在E。杆菌我们发现，当一个Dictyosteelium discoideum的基因序列被放置在Shine-Dalgarno序列的上游，位于启动子和起始密码子之间的靶基因，蛋白质的表达增加。在所检测的基因中，最有效的序列是mlcR，它编码肌球蛋白调节轻链，肌球蛋白II的一个亚基。序列缺失分析表明，lcR基因3′端至少10个碱基的缺失增强了细胞内绿色荧光蛋白的产生。我们将该工具应用于T7表达系统，发现与常规方法相比，测试的蛋白质的表达水平增加。因此，目前的蛋白质生产系统可以通过与TED结合来改进。
Methods for heterologous protein production inEscherichia colihave revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed “translation enhancement by aDictyosteliumgene sequence” (“TED”) inE. coli. We found that protein expression was increased when a gene sequence ofDictyostelium discoideumwas placed upstream of the Shine–Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined wasmlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3′ end of themlcRgene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.