Human SKIP (skeletal muscle and kidney-enriched inositol phosphatase) is a 51 kDa inositol polyphosphate 5’-phosphatase that is found in many tissues including brain, eye, and abundantly in the heart, kidney and skeletal muscle.(1-2) SKIP translocates from the endoplasmic reticulum at rest to the cell membrane upon insulin signaling which stimulates protein complex formation involving the insulin receptor.(3) SKIP exhibits affinity towards the fatty acid phosphatidylinositol 4,5-bisphosphate diC16 (PIP2 or PI(4,5)P2)(4) and it is thought that SKIP hydrolyzes the D-5 position of PIP2.(2) PIP2 is a phospholipid component of cell membranes where it is involved in insulin signaling.(5) The purpose of this experiment was to obtain recombinant, human, enzymatically active SKIP to be used as a tool to study signaling pathways associated with SKIP enzymatic activity. For this purpose, a HIS-SKIP chimera was produced in T7 Shuffle cells. Cultures containing T7 Shuffle cells transformed with pLATE31 SKIP were grown to log phase and protein expression was induced with IPTG (1 mM). Fast protein liquid chromatography (FPLC) using a nickel chromatography column was used to purify HIS-SKIP. HIS-SKIP was detected at 51 kDa using a Western blot using an anti-HIS probe. HIS-SKIP was more pronounced on the Western blot as concentration increased. The total concentration of the non-pure protein mixture which included HIS-SKIP obtained was 1186 µg/ml, as determined by a Bradford assay with a BSA standard curve. The total contribution of HIS-SKIP to the total protein concentration was determined to be ~30% or , ~356 µg/ml as assessed by SDS-PAGE. SKIP enzymatic activity was not detected using the Malachite Green Standard Curve in the partially pure preparation. There may be various reasons for not detecting enzymatic activity.
