Institute of Microbiology and Infection and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Institute of Microbiology and Infection and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
N Biotechnol. 2021 Sep 25;64:1-8. doi: 10.1016/j.nbt.2021.05.001. Epub 2021 May 10.
Many commonly used bacterial promoters employed for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression. However, such promoter systems are often too strong, being ill suited for expressing proteins that are difficult to fold, targeted to the membrane or secreted out of the cytoplasm. To circumvent this problem, a suite of bacterial promoters has been constructed with a range of different promoter strengths, assigning them specific "promoter activity ratings" (PARs). Selecting three of these PAR promoters, with low, intermediate and high strengths, it is demonstrated that the expression of target proteins, such as green fluorescent protein (GFP), human growth hormone (hGH) and single chain variable region antibody fragments (scFvs), can be set to three levels when expressed in E. coli. It is shown that the PAR promoter system is extremely flexible, operating in a variety of E. coli strains and under various different culture regimes. Furthermore, due to its tight regulation, it is shown that this system can also express a toxic outer membrane protein, at levels which do not affect bacterial growth. Thus, the PAR promoter system can be used to tailor the expression levels of target proteins in E. coli and maximize RPP.
许多常用于大肠杆菌中重组蛋白生产（RPP）的常用细菌启动子能够实现高水平的蛋白表达。然而，这样的启动子系统通常过于强大，不适合表达难以折叠、靶向膜或分泌到细胞质外的蛋白。为了解决这个问题，构建了一系列具有不同启动子强度的细菌启动子，并为它们分配了特定的“启动子活性评级”（PAR）。选择这三个 PAR 启动子中的三个，低、中、高强度，结果表明，当在大肠杆菌中表达时，目标蛋白（如绿色荧光蛋白（GFP）、人生长激素（hGH）和单链可变区抗体片段（scFv））的表达可以设定为三个水平。结果表明，PAR 启动子系统非常灵活，在多种大肠杆菌菌株和各种不同的培养条件下都能发挥作用。此外，由于其严格的调控，该系统还可以表达毒性外膜蛋白，而不会影响细菌生长。因此，PAR 启动子系统可用于调整大肠杆菌中目标蛋白的表达水平，从而实现最大的 RPP。
