Defining Membrane Protein Topology Using pho-lac Reporter Fusions
Defining Membrane Protein Topology Using pho-lac Reporter Fusions
复制标题
DOI：
10.1007/978-1-4939-7033-9_10
复制
发表时间：
2017-01-01
期刊：
影响因子：
--
通讯作者：
Ladant, Daniel
中科院分区：
文献类型：
--
作者：
Karimova, Gouzel;Ladant, Daniel
膜蛋白拓扑结构的实验测定可以使用各种技术来实现。在这里，我们提出了pho-lac双报告系统，一个简单，方便，可靠的工具来分析在体内的膜蛋白的拓扑结构。该系统基于使用两种具有互补性质的拓扑标记，即在细胞质中具有活性的大肠杆菌β-半乳糖苷酶LacZ和在细胞质中具有活性的大肠杆菌β-半乳糖苷酶LacZ。coli碱性磷酸酶PhoA，其在细菌周质中具有活性。具体地，在该pho-lac基因系统中，报告分子是由成熟的PhoA组成的嵌合体，其与β-半乳糖苷酶α-肽LacZ α在框内。因此，当靶向周质时，PhoA-LacZ α双报告基因显示出高碱性磷酸酶活性，但没有β-半乳糖苷酶活性。相反，当位于细胞质中时，PhoA-LacZ alpha在大肠杆菌中没有磷酸酶活性，但表现出高的β-半乳糖苷酶活性。表达LacZ的ω片段LacZ ω（通过α-互补现象）的大肠杆菌细胞。PhoA-LacZ α报告基因的双重性质允许一种简单的方法来归一化两种酶活性，以获得关于所研究的膜蛋白和报告基因之间的融合位点的亚细胞位置的易于解释的信息。此外，PhoA-LacZ α报告基因允许利用双指示剂琼脂平板来容易地区分携带细胞质融合、周质融合或框外融合的菌落。总的来说，phoA-lacZ α融合报告方法是一种简单且相当便宜的方法，用于表征体内膜蛋白的拓扑结构。
Experimental determination of membrane protein topology can be achieved using various techniques. Here we present the pho-lac dual reporter system, a simple, convenient, and reliable tool to analyze the topology of membrane proteins in vivo. The system is based on the use of two topological markers with complementary properties, the Escherichia coli beta-galactosidase LacZ, which is active in the cytoplasm, and the E. coli alkaline phosphatase PhoA, which is active in the bacterial periplasm. Specifically, in this pho-lac gene system, the reporter molecule is a chimera composed of the mature PhoA that is in frame with the beta-galactosidase a-peptide, LacZ alpha. Hence, when targeted to the periplasm, the PhoA-LacZ alpha dual reporter displays high alkaline phosphatase activity but no beta-galactosidase activity. Conversely, when located in the cytoplasm, PhoA-LacZ alpha has no phosphatase activity but exhibits high beta-galactosidase activity in E. coli cells expressing the omega fragment of LacZ, LacZ omega (via the alpha-complementation phenomenon). The dual nature of the PhoA-LacZ alpha reporter allows a simple way to normalize both enzymatic activities to obtain readily interpretable information about the subcellular location of the fusion site between the membrane protein under study and the reporter. In addition, the PhoA-LacZ alpha reporter permits utilization of dual-indicator agar plates to easily discriminate between colonies bearing cytoplasmic fusions, periplasmic fusions, or out-of-frame fusions. In total, the phoA-lacZ alpha fusion reporter approach is a straightforward and rather inexpensive method of characterizing the topology of membrane proteins in vivo.