SpringerProtocols: Abstract: 1. Purification of Recombinant G Protein α Subunits from Escherichia coli

1. Purification of Recombinant G Protein α Subunits from Escherichia coli

By: Wendy K. Greentree², Maurine E. Linder²

Abstract

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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 Gα are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein βγ subunits. Methods are described for the expression of G_iα and G_sα 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 Gα 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 Gα are presented.

Affiliation(s): (2) Department of Cell Biology and Physiology, Washington University, St. Louis, MO

Book Title: G Protein Signaling: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 237 | Pub. Date: Sep-15-2003 | Page Range: 3-20 | DOI: 10.1385/1-59259-430-1:3

Subject: Protein Science

Key Words: G protein - α subunit - signal transduction - protein purification - affinity chromatography - GTPase - membrane protein - myristoylation - N-myristoyltransferase

References

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