Inositol polyphosphates are the most widespread second messenger molecules in eukaryotic cells. Human Type I inositol 1,4,5-tri-phosphate (Ins(1,4,5)P-3) 5-phosphatase removes the D-5 position phosphate from soluble Ins(1,4,5)P3, a key event in cell signaling particularly in Ca2+ 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)P3 5-phosphatase is active and can thus be used for functional and structural studies. (C) 2007 Elsevier Inc. All rights reserved.