Kellosalo J, Kajander T, Palmgren M G, Lopéz-Marqués R L, Goldman A
Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Protein Expr Purif. 2011 Sep;79(1):25-34. doi: 10.1016/j.pep.2011.05.020. Epub 2011 Jun 2.
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.
膜结合焦磷酸酶（M-PPases）是一类将无机焦磷酸水解与质子或钠离子泵出相偶联的酶。在植物和细菌中，它们对于缓解缺氧、干旱、营养缺乏、寒冷和低光照强度期间低能量水平所引起的胁迫至关重要。虽然它们在哺乳动物中完全不存在，但它们是致病原生动物生存的关键因素，这使得这些蛋白质成为有吸引力的药理学靶点。在这项工作中，我们旨在纯化出适合结晶的M-PPases，作为获取药物设计结构信息的第一步。我们测试了八种整合膜焦磷酸酶在酿酒酵母中的表达，其中六种来自细菌和古菌来源，两种来自原生动物。两种源自嗜热生物的蛋白质以二聚体和单分散活性状态被纯化出来。为了生成具有增加的亲水性表面积的M-PPases，这可能有助于形成晶体接触，将噬菌体T4溶菌酶插入其中一种M-PPases的不同膜外环中。其中两种融合蛋白具有活性，并且表达水平足以使其纯化用于结晶目的。
