Poly G tails prolong mRNA chemical and functional half life in
E. coli
cell extracts and dramatically increased RNA-dependent protein synthesis in vitro. The effect of polyguanylation on mRNA functional half life, as measured by the ability of CAT transcripts to produce biochemically-active protein in vitro, was four- to six-fold greater than the effect on chemical half life. Addition of a few nucleotides 5′ to the bacteriophage T7 promoter markedly enhanced transcription of linear PCR-generated DNA molecules by T7 RNA polymerase. Collectively a novel approach is provided for efficient in vitro protein synthesis that bypasses the need for cloned DNA templates to obtain the products of translational open reading frames.
聚 G 尾延长大肠杆菌中 mRNA 的
化学半衰期和功能半衰期
大肠杆菌
细胞
提取物中延长了 mRNA 的
化学半衰期和功能半衰期,并显著增加了体外 RNA 依赖性蛋白质合成。多聚
鸟苷酸化对 mRNA 功能半衰期的影响(以 CAT 转录本在体外产生具有
生物化学活性的蛋白质的能力来衡量)是对
化学半衰期影响的四至六倍。在噬菌体 T7 启动子上添加几个核苷酸 5′,可明显增强 T7 RNA 聚合酶对 PCR 生成的线性 DNA 分子的转录。总之,这提供了一种高效体外蛋白质合成的新方法,它不需要克隆 DNA 模板就能获得翻译开放阅读框的产物。