Comparative analysis and “expression space” coverage of the production of prokaryotic membrane proteins for structural genomics
Comparative analysis and “expression space” coverage of the production of prokaryotic membrane proteins for structural genomics
复制标题
用于结构基因组学的原核膜蛋白生产的比较分析和“表达空间”覆盖
作者：
S. Surade;M. Klein;Peggy Stolt;Cornelia Muenke;Ankita Roy;H. Michel
膜蛋白占原核生物和真核生物基因组的三分之一，但已知的膜蛋白结构数量很少。膜蛋白是结构生物学具有挑战性的目标，主要是由于难以生产和纯化毫克量的这些蛋白质。我们正在评估不同的方法来生产和纯化大量的原核细胞膜蛋白，用于随后的结构和功能分析。在这里，我们提出了37个目标蛋白的比较表达数据，所有这些二级转运蛋白，从嗜温生物鼠伤寒沙门氏菌和两个超嗜热生物风产液菌和激烈的热球菌在三个不同的大肠杆菌表达载体。此外，我们研究了使用乳酸乳球菌作为宿主的完整膜蛋白表达。总体而言，78%的目标是在至少一组条件下成功生产的。对这些结果的分析使我们能够评估不同变量在增加我们的目标集的“表达空间”覆盖率中的作用。该分析表明，为了使表达的非同源靶标的数量最大化，应使用C-末端标签在具有不同类型启动子的两种载体中选择并测试直链靶标。此外，E.大肠杆菌是一种表达原核转运蛋白的稳定宿主，其表达能力上级于L.乳。因此，这些结果提示了膜蛋白的高通量异源过量生产的适当策略。
Membrane proteins comprise up to one‐third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing “expression space” coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C‐terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high‐throughput heterologous overproduction of membrane proteins.
DOI：
10.1073/pnas.94.15.8168
发表时间：
1997-07-22
影响因子：
11.1
作者：
Siegele, DA;Hu, JC
通讯作者：
Hu, JC
影响因子：
2.9
作者：
Kiefer, H;Krieger, J;Breer, H
通讯作者：
Breer, H