Expression and characterization of a pleckstrin homology domain in phospholipase C, PLC-η1

Abstract Phospholipase C (PLC) plays an important role in intracellular signal transduction by hydrolyzing phosphatidylinositol 4,5-bis-phosphate, a membrane phospholipid. Currently, thirteen mammalian PLC isozymes have been identified, which are divided into six classes on the basis of structure and mechanisms. All the PLC isozymes share common domains including catalytic X and Y domains, protein kinase C conserved region 2 (C2) domain, EF-hand motif and pleckstrin homology (PH) domain. In this study, the PLC-η1 PH domain has been over-expressed and purified. The most undesirable feature of the protein was instability, resulting in precipitation during the purification process. With the aim of structural characterization, a solution condition was optimized using SDS–PAGE and NMR spectroscopy. A circular dichroism spectrum indicated that the PLC-η1 PH domain mainly comprised β-strands, which was also suggested by the 2D 1 H– 15 N HSQC spectrum.