title: Heterologous expression and purification of membrane-bound pyrophosphatases.
abstract: Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low light intensity. While they are completely absent in mammalians, they are key players in the survival of disease-causing protozoans making these proteins attractive pharmacological targets. In this work, we aimed at the purification of M-PPases in amounts suitable for crystallization as a first step to obtain structural information for drug design. We have tested the expression of eight integral membrane pyrophosphatases in Saccharomyces cerevisiae, six from bacterial and archaeal sources and two from protozoa. Two proteins originating from hyperthermophilic organisms were purified in dimeric and monodisperse active states. To generate M-PPases with an increased hydrophilic surface area, which potentially should facilitate formation of crystal contacts, phage T4 lysozyme was inserted into different extramembraneous loops of one of these M-PPases. Two of these fusion proteins were active and expressed at levels that would allow their purification for crystallization purposes.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: J
authors.initials: J
authors.lastname: Kellosalo
authors.forename: T
authors.initials: T
authors.lastname: Kajander
authors.forename: M G
authors.initials: MG
authors.lastname: Palmgren
authors.forename: R L
authors.initials: RL
authors.lastname: Lopéz-Marqués
authors.forename: A
authors.initials: A
authors.lastname: Goldman
mesh_terms: D001105:Archaea / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification*; D017122:Bacteriophage T4 / Q000201:enzymology; D003001:Cloning, Molecular; D015870:Gene Expression; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification*; D009113:Muramidase / Q000235:genetics / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification; D041543:Pyrobaculum / Q000201:enzymology / Q000235:genetics; D011755:Pyrophosphatases / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D009113:Muramidase; D011755:Pyrophosphatases