Defining Membrane Protein Topology Using pho-lac Reporter Fusions
| Autor: | Gouzel Karimova, Daniel Ladant |
|---|---|
| Přispěvatelé: | Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Institut Pasteur and Centre National de la Recherche Scientifique (CNRS UMR 3528, Biologie Structurale et Agents Infectieux)., Laure Journet, Eric Cascales, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
MESH: Enzyme Activation MESH: Gene Expression [SDV]Life Sciences [q-bio] Phosphatase lac operon β-galactosidase MESH: Escherichia coli / genetics MESH: Membrane Proteins / genetics Topology medicine.disease_cause MESH: Gene Order MESH: Membrane Proteins / chemistry 03 medical and health sciences Membrane proteins medicine MESH: Cloning Molecular MESH: Recombinant Fusion Proteins Escherichia coli Chemistry MESH: Genes Reporter Dual reporter system MESH: Plasmids / genetics [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry Molecular Biology/Molecular biology Periplasmic space MESH: Lac Operon [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology 030104 developmental biology Membrane protein Cytoplasm Membrane topology Alkaline phosphatase bacteria MESH: Escherichia coli / metabolism MESH: beta-Galactosidase / genetics |
| Zdroj: | Bacterial Protein Secretion Systems Laure Journet; Eric Cascales. Bacterial Protein Secretion Systems, 1615, Humana Press; Springer Science, pp.129-142, 2017, Methods in Molecular Biology, 978-1-4939-7033-9. ⟨10.1007/978-1-4939-7033-9_10⟩ Methods in Molecular Biology ISBN: 9781493970315 |
| DOI: | 10.1007/978-1-4939-7033-9_10⟩ |
| Popis: | International audience; 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 β-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 β-galactosidase α-peptide, LacZα. Hence, when targeted to the periplasm, the PhoA-LacZα dual reporter displays high alkaline phosphatase activity but no β-galactosidase activity. Conversely, when located in the cytoplasm, PhoA-LacZα has no phosphatase activity but exhibits high β-galactosidase activity in E. coli cells expressing the ω fragment of LacZ, LacZω (via the α-complementation phenomenon). The dual nature of the PhoA- LacZα 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-LacZα 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-lacZα fusion reporter approach is a straightforward and rather inexpensive method of characterizing the topology of membrane proteins in vivo. |
| Databáze: | OpenAIRE |
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