|
rdf:type |
|
|
lifeskim:mentions |
|
|
pubmed:issue |
1
|
|
pubmed:dateCreated |
2005-9-26
|
|
pubmed:abstractText |
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.
|
|
pubmed:language |
eng
|
|
pubmed:journal |
|
|
pubmed:citationSubset |
IM
|
|
pubmed:chemical |
|
|
pubmed:status |
MEDLINE
|
|
pubmed:month |
Oct
|
|
pubmed:issn |
0006-3002
|
|
pubmed:author |
|
|
pubmed:issnType |
Print
|
|
pubmed:day |
1
|
|
pubmed:volume |
1716
|
|
pubmed:owner |
NLM
|
|
pubmed:authorsComplete |
Y
|
|
pubmed:pagination |
69-76
|
|
pubmed:dateRevised |
2006-11-15
|
|
pubmed:meshHeading |
pubmed-meshheading:16182234-Blotting, Western,
pubmed-meshheading:16182234-Cell Membrane,
pubmed-meshheading:16182234-Chromatography, Affinity,
pubmed-meshheading:16182234-Detergents,
pubmed-meshheading:16182234-Dimerization,
pubmed-meshheading:16182234-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:16182234-Hydrogen-Ion Concentration,
pubmed-meshheading:16182234-Image Processing, Computer-Assisted,
pubmed-meshheading:16182234-Inorganic Pyrophosphatase,
pubmed-meshheading:16182234-Lipids,
pubmed-meshheading:16182234-Membrane Proteins,
pubmed-meshheading:16182234-Microscopy, Electron,
pubmed-meshheading:16182234-Mutagenesis,
pubmed-meshheading:16182234-Nickel,
pubmed-meshheading:16182234-Plasmids,
pubmed-meshheading:16182234-Protons,
pubmed-meshheading:16182234-Recombinant Proteins,
pubmed-meshheading:16182234-Saccharomyces cerevisiae,
pubmed-meshheading:16182234-Structure-Activity Relationship,
pubmed-meshheading:16182234-Temperature,
pubmed-meshheading:16182234-Thermotoga maritima
|
|
pubmed:year |
2005
|
|
pubmed:articleTitle |
Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: a "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins.
|
|
pubmed:affiliation |
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla, CSIC, Avda. Americo Vespucio 49, 45092 Sevilla, Spain. rlo@kvl.dk
|
|
pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
|
