The Gram-negative bacterium Escherichia coli has been the work horse for recombinant protein production since the past several years. However, most of the gene expression systems used either require expensive inducers or exhibit low strength. In the present study, we have generated a strong promoter by repeated rounds of random mutagenesis in a stationary phase promoter isolated from Gordonia sp. IITR100. The promoter activity increased 16-fold as compared to the wild-type promoter. The resultant synthetic promoter showed β-galactosidase activities of ~16,000 Miller units which is comparable to the strong T7 promoter ~13,000 Miller units. The amount of LacZ produced by the synthetic promoter was found to be active for several days in stationary phase. The advantage of this synthetic promoter over T7 promoter includes its stationary phase auto-inducibility thereby saving the cost of addition of inducers. Expression of GFPuv was observed in all the cells of E. coli due to the absence of requirement of inducer. A general-purpose vector containing the synthetic promoter with an MCS ready for use has been developed in the study. It has also been used to demonstrate the production of two heterologous proteins.

中文翻译：

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基于强合成静止期启动子的大肠杆菌基因表达系统。

自过去几年以来，革兰氏阴性细菌大肠埃希氏菌一直是重组蛋白生产的主力军。然而，大多数使用的基因表达系统要么需要昂贵的诱导剂，要么表现出低强度。在本研究中，我们通过从Gordonia sp。分离的固定相启动子中反复进行随机诱变产生了强大的启动子。IITR100。与野生型启动子相比，启动子活性增加了16倍。所得的合成启动子显示约16,000 Miller单位的β-半乳糖苷酶活性，与约13,000 Miller单位的强T7启动子相当。发现由合成启动子产生的LacZ的量在固定相中活跃了几天。该合成启动子优于T7启动子的优点包括其固定相自诱导性，从而节省了添加诱导剂的成本。由于不需要诱导剂，在大肠杆菌的所有细胞中均观察到GFPuv的表达。该研究中已经开发了一种通用载体，该载体包含带有MCS的合成启动子。它也已用于证明两种异源蛋白的产生。