Genetic analysis of membrane protein topology by a sandwich gene fusion approach.

We describe a cloning vector that allows the construction of phoA sandwich fusions in which mature alkaline phosphatase is inserted into target proteins. In contrast to previous fusions obtained using the TnphoA transposon, the entire amino acid sequence of the target protein is present in the fusion product. We have constructed a series of sandwich fusions of alkaline phosphatase to the multispanning cytoplasmic membrane protein MalF. Despite the fact that the alkaline phosphatase was tethered to MalF at both its N and its C terminus, the enzyme exhibited high activity when it was fused to a periplasmic domain of the membrane protein. Cells harboring an alkaline phosphatase sandwich fusion to the end of the first membrane-spanning segment of MalF exhibited both MalF and alkaline phosphatase activity. When alkaline phosphatase was inserted into a cytoplasmic domain of MalF, its specific activity was very low. Our results suggest that the alkaline phosphatase activity of phoA sandwich fusions provides a more sensitive monitor than previous methods of the cellular localization of the domain of the target protein to which the enzyme is fused. Thus, the sandwich fusion approach can give a more accurate picture of membrane protein topology. IMAGES:

本研究描述了一种克隆载体，可用于构建phoA三明治融合体（phoA sandwich fusion），即将成熟碱性磷酸酶（alkaline phosphatase）插入靶蛋白内部。相较于此前利用TnphoA转座子（TnphoA transposon）构建的融合体系，该融合产物可完整保留靶蛋白的全部氨基酸序列。我们构建了一系列碱性磷酸酶与多次跨膜细胞质膜蛋白MalF的三明治融合体。尽管碱性磷酸酶的N端与C端均与MalF相连，但当该酶融合至膜蛋白的周质结构域时，仍可表现出较高的酶活性。携带MalF首个跨膜区段末端碱性磷酸酶三明治融合体的宿主细胞，可同时表现出MalF与碱性磷酸酶活性。若将碱性磷酸酶插入MalF的胞质结构域，则其比活性极低。本研究结果表明，phoA三明治融合体的碱性磷酸酶活性可较传统方法更灵敏地监测酶所融合的靶蛋白结构域的细胞定位情况。因此，三明治融合策略可更为准确地解析膜蛋白的拓扑结构。附图：