Genetic analysis of membrane protein topology by a sandwich gene fusion approach.
AUTOR(ES)
Ehrmann, M
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=54790Documentos Relacionados
- Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.
- Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.
- Membrane topology model of Escherichia coli alpha-ketoglutarate permease by phoA fusion analysis.
- Dilated cardiomyopathy: a genetic approach.
- Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis