Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.
Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.
复制标题
使用 T4::T7-RNAP 噬菌体表达包装处理系统防止蛋白水解。
DOI：
10.1016/0378-1119(95)00327-3
复制
发表时间：
1995
期刊：
影响因子：
3.5
通讯作者：
Black,LW
中科院分区：
文献类型：
--
作者：
Hong,YR;Mullaney,JM;Black,LW
将编码噬菌体T7 RNA聚合酶（T7-RNAP）的DNA插入T4基因组的阳性选择载体形式中，将其置于噬菌体T4 ipIII启动子的控制下。重组T4：：T7-RNAP融合噬菌体保留感染性，并在感染的细胞中产生T7-RNAP。通过插入到含有iPIII（编码内部蛋白III）靶部分和噬菌体T7启动子区的质粒中构建融合基因。当用T4：：T7-RNAP re-phage感染含有质粒的大肠杆菌细胞时，细菌产生高水平的融合蛋白。新合成的T4：：T7-RNAP重噬菌体子代在头部形态发生期间将融合蛋白包装并加工到噬菌体衣壳中。本论文中我们证明了截短的T4内部蛋白IPIII、人IPIII：：βGlo（β-globin）融合蛋白、E.大肠杆菌IPIII：：βGlo：：βGal（β-半乳糖苷酶）三重融合蛋白和IPIII：：V3融合蛋白（人免疫缺陷病毒包膜蛋白gp 120 V3区）通过T4：：T7-RNAP诱导以高水平表达。使用IPIII：：βGlo，表达包装加工（EPP）与T4：：T7-RNAP再噬菌体感染同时发生。我们还证明了T4：：T7-RNAP重噬菌体稳定不稳定的蛋白质，如βGal的X90片段，认为其被lon蛋白酶降解。人细胞色素b558大亚基的一个不稳定的20-kDa片段是吞噬细胞中的一种完整膜蛋白，即使在离子缺陷型BL 21菌株中产生，也会发生蛋白水解降解。然而，在用T4：：T7-RNAP re-噬菌体诱导后，产生完整的20-kDa蛋白。因此，T4：：T7-RNAP重噬菌体似乎提供了一种简单、快速和通用的方法，以高产率生产蛋白质，将IPIII融合蛋白包装和加工成易于操作的噬菌体衣壳，并保护蛋白质免于蛋白水解。
DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted into a positive selection-vector form of the T4 genome, placing it under the control of bacteriophage T4 ipIII promoters. The recombinant T4::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in infected cells. Fusion genes were constructed by insertion into a plasmid containing an iPIII (encoding internal protein III) target portion and a bacteriophage T7 promoter region. When Escherichia coli cells containing the plasmid were infected with the T4::T7-RNAP re-phage, the bacteria produced fusion protein at high levels. The newly synthesized T4::T7-RNAP re-phage progeny package and process the fusion protein into the phage capsid during head morphogenesis. In this paper, we demonstrate that truncated T4 internal protein IPIII, human IPIII::βGlo (β-globin) fusion protein, E. coli IPIII::βGlo::βGal (β-galactosidase) triple-fusion protein and IPIII::V3 fusion protein (human immunodeficiency virus envelope protein gp120 V3 region) are expressed at high levels by T4::T7-RNAP induction. With IPIII::βGlo, expression-packaging-processing (EPP) occurs simultaneously with T4::T7-RNAP re-phage infection. We also demonstrate that T4::T7-RNAP re-phage stabilize unstable proteins such as the X90 fragment of βGal, thought to be degraded by the lon protease. An unstable 20-kDa fragment of the large subunit of human cytochrome b558an integral membrane protein in phagocytes, is subject to proteolytic degradation even when produced in the lon-deficient BL21 strain. However, upon induction with T4::T7-RNAP re-phage, the 20-kDa protein is produced intact. Thus, T4::T7-RNAP re-phage appear to provide a simple, rapid and universal means of producing proteins in high yield, packaging and processing IPIII fusion proteins into easily manipulated phage capsids, and protecting proteins from proteolysis.