Heterologous expression and purification of membrane-bound pyrophosphatases
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Heterologous expression and purification of membrane-bound pyrophosphatases. / Kellosalo, J.; Kajander, T.; Palmgren, Michael Broberg; Lopez Marques, Rosa Laura; Goldman, A.
In: Protein Expression and Purification, Vol. 79, No. 1, 2011, p. 25-34.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Heterologous expression and purification of membrane-bound pyrophosphatases
AU - Kellosalo, J.
AU - Kajander, T.
AU - Palmgren, Michael Broberg
AU - Lopez Marques, Rosa Laura
AU - Goldman, A.
PY - 2011
Y1 - 2011
N2 - 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. Highlights ¿ 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.
AB - 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. Highlights ¿ 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.
U2 - 10.1016/j.pep.2011.05.020
DO - 10.1016/j.pep.2011.05.020
M3 - Journal article
C2 - 21664973
VL - 79
SP - 25
EP - 34
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -
ID: 33824039