Document Detail

Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: a "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins.
MedLine Citation:
PMID:  16182234     Owner:  NLM     Status:  MEDLINE    
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.
Rosa L López-Marqués; José R Pérez-Castiñeira; Morten J Buch-Pedersen; Sergio Marco; Jean-Louis Rigaud; Michael G Palmgren; Aurelio Serrano
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  1716     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  2005 Oct 
Date Detail:
Created Date:  2005-09-26     Completed Date:  2005-11-21     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  69-76     Citation Subset:  IM    
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla, CSIC, Avda. Americo Vespucio 49, 45092 Sevilla, Spain.
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MeSH Terms
Blotting, Western
Cell Membrane / metabolism
Chromatography, Affinity
Detergents / pharmacology
Electrophoresis, Polyacrylamide Gel
Hydrogen-Ion Concentration
Image Processing, Computer-Assisted
Inorganic Pyrophosphatase / chemistry*,  isolation & purification*
Lipids / chemistry
Membrane Proteins / chemistry
Microscopy, Electron
Nickel / chemistry
Plasmids / metabolism
Recombinant Proteins / chemistry
Saccharomyces cerevisiae / metabolism
Structure-Activity Relationship
Thermotoga maritima / metabolism*
Reg. No./Substance:
0/Detergents; 0/Lipids; 0/Membrane Proteins; 0/Protons; 0/Recombinant Proteins; 7440-02-0/Nickel; EC Pyrophosphatase

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