Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: a "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins.
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.
López-Marqués
Rosa L
RL
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla, CSIC, Avda. Americo Vespucio 49, 45092 Sevilla, Spain. rlo@kvl.dk
Pérez-Castiñeira
José R
JR
Buch-Pedersen
Morten J
MJ
Marco
Sergio
S
Rigaud
Jean-Louis
JL
Palmgren
Michael G
MG
Serrano
Aurelio
A
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000302:isolation & purification*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008854:Microscopy, Electron; D016296:Mutagenesis; D009532:Nickel / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D020124:Thermotoga maritima / Q000378:metabolism*
D003902:Detergents; D008055:Lipids; D008565:Membrane Proteins; D011522:Protons; D011994:Recombinant Proteins; D009532:Nickel; D043564:Inorganic Pyrophosphatase