EXTRACELLULAR EXPORT OF CLONED GENE PRODUCTS

克隆基因产品的细胞外出口

基本信息

批准号：
3291520
负责人：
Michael J Benedik
金额：
$ 11.29万
依托单位：
UNIVERSITY OF HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-07-01 至 1991-06-30
项目状态：
已结题

项目摘要

We propose to develop a system to study the mechanisms of extracellular secretion in enteric bacteria. The genetic approaches used in Escherichia coli to study protein targeting and localization have proven extremely powerful, but E. coli does not normally secrete protein out of the cell. Serratia marcescens does secrete a number of proteins abundantly into the growth media. Being an enterobacteria it is closely related to E. coli and many of the plasmid and phage vectors developed for E. coli will function equally well in S. marcescens, as the exoproteins of S. marcescens will function and be secreted in E. coli. We propose to use secreted exonuclease from S. marcescens as a model to develop secretion systems for genetic study in both E. coli and S. marcescens. We have already identified the exonuclease gene and it is presently being sequenced. We have shown that it is also secreted in E. coli as well as in S. marcescens. By constructing protein fusion between the exoprotein and a marker gene such as alkaline phosphatase or beta-galactosidase, we hope to identify regions involved in protein export. Selection schemes based on the fusion proteins, or based on properties intrinsic to the nuclease, will be used to isolate mutations which alter or abolish extracellular secretion. These mutations could either be within the nuclease gene itself, thus identifying specific signals for secretion, or could be within host genes required for secretion. Mutations affecting protein export in E. coli will be tested to determine how they effect extracellular secretion; this should help characterize the relationship between these processes. Using LacZ fusions which inhibit normal protein export in E. coli when overexpressed, will be introduced into S. marcescens to test their effect on secretion of the exoproteins of S. marcescens. Experiments designed to probe the path of exonuclease secretion are planned. We hope to use variety of approaches to elucidate the interactions between the exonuclease and the host export mechanism and to determine the path of secretion. Competition experiments can determine the common steps in the secretion process. Components which are limiting might be identified by these competition experiments. The long term goals of this research are to understand the mechanism of this one type of extracellular secretion, to compare and contrast extracellular secretion and export of envelope proteins, and to determine if the exoproteins of S. marcescens utilize a unique path for extracellular secretion distinct from the secretion of exoproteins by other gram negative bacteria.

我们建议开发一个系统来研究肠细菌的细胞外分泌。遗传大肠杆菌中使用的方法研究蛋白质靶向和事实证明，本地化非常强大，但大肠杆菌没有通常将蛋白质从细胞中分泌出来。 Serratia Marcescens 会大量分泌多种蛋白质的生长媒体。作为肠杆菌，它与大肠杆菌和大肠杆菌密切相关为大肠杆菌开发的许多质粒和噬菌体载体将在马尔斯霉菌链球菌中，功能同样很好，与S. Marcescens将发挥作用并在大肠杆菌中分泌。我们建议将分泌的外切的分泌的外切酶作为模型开发大肠杆菌和S.遗传研究的分泌系统马尔斯奇斯。我们已经确定了核酸外切酶基因，这是目前正在测序。我们已经证明它也被分泌在大肠杆菌以及链球菌中。通过构造蛋白质异蛋白和标记基因（例如碱性）之间的融合磷酸酶或β-半乳糖苷酶，我们希望识别区域参与蛋白质出口。基于融合蛋白的选择方案或基于核酸酶固有的特性将用于分离改变或废除细胞外分泌的突变。这些突变可以在核酸酶基因本身内，因此识别分泌的特定信号，或者可以在主机中分泌所需的基因。影响蛋白质输出的突变将在大肠杆菌中进行测试以确定它们的影响细胞外分泌；这应该有助于表征这些过程之间的关系。使用lacz融合抑制大肠杆菌中正常蛋白的导出时，将被引入S. Marcescens以测试其对分泌的影响 S. marcescens的脱蛋白。旨在探测外切核酸酶分泌的路径的实验计划。我们希望使用各种方法来阐明外切酶与主机导出之间的相互作用机制并确定分泌路径。竞赛实验可以确定分泌的共同步骤过程。限制的组件可能会通过这些竞争实验。这项研究的长期目标是了解一种类型的细胞外分泌的机制，以比较以及对比细胞外分泌和信封的出口蛋白质，并确定Marcescens的Opropotins是否脱蛋白利用一条独特的路径来与细胞外分泌不同其他革兰氏阴性细菌对摄起蛋白的分泌。