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KMID : 0613820110210071032
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Journal of Life Science
2011 Volume.21 No. 7 p.1032 ~ p.1038
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Expression and Purification of the Phosphatase-like Domain of a Voltage-Sensing Phosphatase, Ci-VSP
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Kim Sung-Jae
Kim Hae-Min
Choi Hoon
Kim Young-Jun
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Abstract
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Recently identified Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) consists of an ion channel-like transmembrane domain (VSD) and a phosphatase-like domain. Ci-VSP senses the change of membrane potential by its VSD and works as a phosphoinositide phosphatase by its phosphatase domain. In this study, we present the construction of His-tagged phosphatase-like domain of Ci-VSP, its recombinant expression and purification, and its enzymatic activity behavior in order to examine the biochemical behavior of phosphatase domain of Ci-VSP without interference. We found that Ci-VSP(248-576)-His can be eluted with an elution buffer containing 25 mM NaCl and 100 mM imidazole during His-tag purification. In addition, we found the proper measurement condition for kinetics study of Ci-VSP(248-576)-His against p-nitrophenyl phosphate (pNPP). We measured the kinetic constant of Ci-VSP(248-576)-His at 37℃, pH 5.0 or 5.5, under 30 min of reaction time, and less than 2.0 ㎍ of protein amount. With these conditions, we acquired that Ci-VSP(248-576)-His has Km of 354±0.143 μM, Vmax of 0.0607±0.0137 μ㏖/min/㎎ and kcat of 0.359±0.009751 min?¹ for pNPP dephosphorylation. Therefore, we produced a pure form of Ci-VSP(248-576)-His, and this showed a higher activity against pNPP. This purified protein will provide the road to a structural investigation on an interesting protein, Ci-VSP.
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KEYWORD
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Ciona intestinalis voltage sensor-containing phosphatase(Ci-VSP), phosphatidyl inositol phosphate, lipid phosphatase, Km, Vmax
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