Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10798110 | Biochimica et Biophysica Acta (BBA) - Biomembranes | 2005 | 8 Pages |
Abstract
Although several proton-pumping pyrophosphatases (H+-PPases) have been overexpressed in heterologous systems, purification of these recombinant integral membrane proteins in large amounts in order to study their structure-function relationships has proven to be a very difficult task. In this study we report a new method for large-scale production of pure and stable thermophilic H+-PPase from Thermotoga maritima. Following overexpression in yeast, a “Hot-Solve” procedure based on high-temperature solubilization and metal-affinity chromatography was used to obtain a highly purified detergent-solubilized TVP fraction with a yield around 1.5 mg of protein per litre of yeast culture. Electron microscopy showed the monodispersity of the purified protein and single particle analysis provided the first direct evidence of a dimeric structure for H+-PPases. We propose that the method developed could be useful for large-scale purification of other recombinant thermophilic membrane proteins.
Keywords
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Authors
Rosa L. López-Marqués, José R. Pérez-Castiñeira, Morten J. Buch-Pedersen, Sergio Marco, Jean-Louis Rigaud, Michael G. Palmgren, Aurelio Serrano,