کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020839 1069213 2011 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Heterologous expression and purification of membrane-bound pyrophosphatases
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Heterologous expression and purification of membrane-bound pyrophosphatases
چکیده انگلیسی

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.


► We have tested expression of eight membrane-bound pyrophosphatases in Saccharomyces cerevisiae.
► We have made functional T4-lysozyme-membrane-bound pyrophosphatase chimeras.
► We have purified membrane-bound pyrophosphatase of Pyrobaculum aerophilum.
► We show that membrane-bound pyrophosphatases of Pyrobaculum aerophilum and Thermotoga maritima are purified in dimeric form.
► Activities of P. aerophilum and T. maritima membrane-bound pyrophosphatases tested in different detergents.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 79, Issue 1, September 2011, Pages 25–34
نویسندگان
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