Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: a "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins

Biochim Biophys Acta. 2005 Oct 1;1716(1):69-76. doi: 10.1016/j.bbamem.2005.08.004.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Membrane / metabolism
  • Chromatography, Affinity
  • Detergents / pharmacology
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen-Ion Concentration
  • Image Processing, Computer-Assisted
  • Inorganic Pyrophosphatase / chemistry*
  • Inorganic Pyrophosphatase / isolation & purification*
  • Lipids / chemistry
  • Membrane Proteins / chemistry
  • Microscopy, Electron
  • Mutagenesis
  • Nickel / chemistry
  • Plasmids / metabolism
  • Protons*
  • Recombinant Proteins / chemistry
  • Saccharomyces cerevisiae / metabolism
  • Structure-Activity Relationship
  • Temperature
  • Thermotoga maritima / metabolism*


  • Detergents
  • Lipids
  • Membrane Proteins
  • Protons
  • Recombinant Proteins
  • Nickel
  • Inorganic Pyrophosphatase