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Purification of Phospholipase C β and Phospholipase C ε from Sf9 Cells

descriptionPublicationkeyboard_double_arrow_right Part of book or chapter of book , Article 15 Nov 2003Publisher:Humana Press

Authors: Huan Wang; Mousumi Ghosh; Grant G. Kelley; Alan V. Smrcka;

doi: 10.1385/1-59259-430-1:55

pmid: 14501038

Purification of Phospholipase C β and Phospholipase C ε from Sf9 Cells

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Abstract

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP(3)). The PLC beta isoforms of PLCs are activated by G proteins after hormone or neurotransmitter stimulation of G protein-coupled receptors (GPCR). PLC epsilon is a recently identified PLC isoform that is activated by Ras and G beta gamma subunit although the physiological role of this enzyme is not well understood. Methods for purification of PLC beta and PLC epsilon from Sf9 cells are described. In the case of hexahistidine (6-His)-tagged PLC beta the purification involves two steps, affinity chromatography with Ni-NTA agarose followed by heparin Sepharose chromatography. 6-His-tagged PLC epsilon can be purified in a single step with nickel nitrilotriacetic acid-agarose (Ni-NTA) affinity chromatography.

Related Organizations

University of Rochester
United States
SUNY Upstate Medical University
United States

Keywords

Insecta, Phospholipase C beta, Chromatography, Affinity, Recombinant Proteins, Cell Line, Isoenzymes, Phosphoinositide Phospholipase C, Type C Phospholipases, Animals, Signal Transduction

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