Expression and characterization of a pleckstrin homology domain in phospholipase C, PLC-eta1.
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-eta1 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-eta1 PH domain mainly comprised beta-strands, which was also suggested by the 2D 1H-15N HSQC spectrum.
Imai
Toru
T
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Kasai
Kouki
K
Kurita
Junichi
J
Fukami
Kiyoko
K
Tashiro
Mitsuru
M
Shimotakahara
Sakurako
S
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D054801:Phosphoinositide Phospholipase C / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D010738:Type C Phospholipases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D001798:Blood Proteins; D007527:Isoenzymes; D010750:Phosphoproteins; C039077:platelet protein P47; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C506061:Plch1 protein, mouse