Wohlkönig Alexandre, Sénéchal Magalie, Dewitte Frédérique, Backers Katrien, Erneux Christophe, Villeret Vincent
UMR8161, Institut de Biologie de Lille, CNRS/Université de Lille1/Université de Lille2/Institut Pasteur de Lille, IFR 142, 1 rue du Professeur Calmette, Lille 59021, France.
Protein Expr Purif. 2007 Sep;55(1):69-74. doi: 10.1016/j.pep.2007.04.009. Epub 2007 Apr 24.
Inositol polyphosphates are the most widespread second messenger molecules in eukaryotic cells. Human Type I inositol 1,4,5-triphosphate (Ins(1,4,5)P(3)) 5-phosphatase removes the D-5 position phosphate from soluble Ins(1,4,5)P(3,) a key event in cell signaling particularly in Ca(2+) homeostasis. In this study, the cDNA encoding human Type I Ins(1,4,5)P(3) 5-phosphatase was subcloned into a modified pMAL expression vector. This plasmid produces a recombinant protein in fusion with affinity tags located at its N-terminus, consisting in a maltose binding protein (MPB) and an octa-histidine stretch. The construction was transformed into Escherichia coli BL21 (DE3) expression strain. This dual tag strategy allows the purification of milligrams of highly purified protein. The recombinant human Type I Ins(1,4,5)P(3) 5-phosphatase is active and can thus be used for functional and structural studies.
肌醇多磷酸是真核细胞中分布最广泛的第二信使分子。人I型肌醇1,4,5-三磷酸（Ins(1,4,5)P(3)）5-磷酸酶从可溶性Ins(1,4,5)P(3)中去除D-5位磷酸，这是细胞信号传导中的关键事件，特别是在Ca(2+)稳态中。在本研究中，将编码人I型Ins(1,4,5)P(3) 5-磷酸酶的cDNA亚克隆到修饰的pMAL表达载体中。该质粒产生与位于其N端的亲和标签融合的重组蛋白，该亲和标签由麦芽糖结合蛋白（MPB）和八聚组氨酸序列组成。构建体被转化到大肠杆菌BL21(DE3)表达菌株中。这种双标签策略能够纯化出毫克级的高度纯化蛋白。重组人I型Ins(1,4,5)P(3) 5-磷酸酶具有活性，因此可用于功能和结构研究。
