Expression and purification in high yield of a functionally active recombinant human Type I inositol(1,4,5)P3 5-phosphatase.
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.
Wohlkönig
Alexandre
A
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. alexandre.wohlkonig@ibl.fr
Sénéchal
Magalie
M
Dewitte
Frédérique
F
Backers
Katrien
K
Erneux
Christophe
C
Villeret
Vincent
V
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D058619:Maltose-Binding Proteins; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases