Snapyan Marina, Robin Sylvain, Yeretssian Garabet, Lecocq Michèle, Marc Frédéric, Sakanyan Vehary
UMR CNRS 6204, Faculté des Sciences et des Techniques, Université de Nantes, 44322 Nantes, France.
ProtNeteomix, 44322 Nantes, France.
BioTech (Basel). 2021 Oct 14;10(4):24. doi: 10.3390/biotech10040024.
We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong p promoter, originally isolated from a moderate thermophilic bacterium , was used to improve the performance of a cell-free system in extracts of BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the RNA polymerase subunits α, β, β' and ω are simultaneously coexpressed. Appending a 3' UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The BL21 (DE3) ::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial p and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium , synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.
我们评估了几种在无细胞偶联细菌转录和翻译系统中提高蛋白质合成的方法。一个最初从嗜温细菌中分离出的强p启动子，被用于提高无细胞系统在BL21(DE3)提取物中的性能。在重组细胞制备的提取物中检测到对蛋白质合成的刺激作用，其中RNA聚合酶亚基α、β、β'和ω同时共表达。在蛋白质编码区附加一个3'UTR基因组序列和一个T7转录终止子，也能提高一些线性DNA基因的合成活性。构建了周质RNase I缺陷的BL21(DE3)::Tn10突变体。该突变体的无细胞提取物使细菌p启动子和噬菌体pT7启动子介导的细菌和人类基因表达提高了四倍。相比之下，RNase E缺陷并不影响相同基因的无细胞表达。在无细胞系统中合成的嗜极细菌调节蛋白，能够提供与目标DNA区域的结合能力。所描述的无细胞系统的有利特性为高通量筛选分析提供了诱人的机会。
