Ghosh Mousumi, Wang Huan, Kelley Grant G, Smrcka Alan V
Department of Pharmacology and Physiology, University of Rochester School of Medicine, NY, USA.
Methods Mol Biol. 2004;237:55-64. doi: 10.1385/1-59259-430-1:55.
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.
磷脂酰肌醇特异性磷脂酶C（PLC）催化磷脂酰肌醇4,5-二磷酸（PIP2）水解，生成二酰基甘油（DAG）和肌醇1,4,5-三磷酸（IP(3)）。激素或神经递质刺激G蛋白偶联受体（GPCR）后，PLC的PLCβ亚型会被G蛋白激活。PLCε是最近发现的一种PLC亚型，它可被Ras和Gβγ亚基激活，不过这种酶的生理作用尚未完全明确。本文介绍了从Sf9细胞中纯化PLCβ和PLCε的方法。对于带有六组氨酸（6-His）标签的PLCβ，纯化过程包括两个步骤，先用Ni-NTA琼脂糖进行亲和层析，然后用肝素琼脂糖层析。带有6-His标签的PLCε可以通过镍次氮基三乙酸-琼脂糖（Ni-NTA）亲和层析一步纯化。
