Greentree Wendy K, Linder Maurine E
Department of Cell Biology and Physiology, Washington University, St. Louis, MO, USA.
Methods Mol Biol. 2004;237:3-20. doi: 10.1385/1-59259-430-1:3.
The purification of recombinant G protein a subunits expressed in Escherichia coli (E. coli) is a convenient and inexpensive method to obtain homogeneous preparations of protein for biochemical and biophysical analyses. Wild-type and mutant forms of G alpha are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein beta gamma subunits. Methods are described for the expression of Gi alpha and Gs alpha proteins in E. coli. Protocols are provided for the purification of untagged G protein a subunits using conventional chromatography and histidine (His)-tagged subunits using metal chelate chromatography. Modification of G alpha with myristate can be recapitulated in E. coli by expressing N-myristoyltransferase (NMT) with its G protein substrate. Protocols for the production and purification of myristoylated G alpha are presented.
在大肠杆菌（E. coli）中表达的重组G蛋白α亚基的纯化是一种方便且廉价的方法，可获得用于生化和生物物理分析的蛋白质纯品。Gα的野生型和突变形式很容易制备，用于分析其内在生化特性，以及与受体、效应器、调节剂和G蛋白βγ亚基进行重组。本文描述了在大肠杆菌中表达Giα和Gsα蛋白的方法。提供了使用常规色谱法纯化无标签G蛋白α亚基以及使用金属螯合色谱法纯化组氨酸（His）标签亚基的方案。通过与G蛋白底物一起表达N-肉豆蔻酰转移酶（NMT），可在大肠杆菌中重现Gα与肉豆蔻酸的修饰。本文还介绍了肉豆蔻酰化Gα的生产和纯化方案。
