Expression, purification and characterisation of a functional phosphatidylinositol-specific phospholipase C-delta 1 protein in Escherichia coli.
Inositol-lipid-specific phospholipase C-delta 1 (PtdIns-PLC delta 1) was expressed in Escherichia coli as a fusion protein containing a short 22-amino-acid lac-Z-derived amino terminus. Under appropriate conditions, the phospholipase constituted approximately 0.2% of the detergent-soluble protein and could be purified to near homogeneity in a simple three step protocol. The catalytic properties of the purified enzyme closely resemble those of the eukaryote-derived protein. The suitability of bacterial expression for the investigation of PtdIns-PLC delta regulation is discussed.
Ginger
R S
RS
Imperial Cancer Research Fund, London, England.
Parker
P J
PJ
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D007527:Isoenzymes; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium