This chapter describes a general method for expressing several G-protein α subunits in Escherichia coli (E. coli) at levels 10-100 times higher than achieved previously. G proteins are a family of guanine nucleotide-binding regulatory proteins that link a large number of cell surface receptors to regulation of several intracellular effectors. The chapter describes a method for purification of the recombinant proteins by affinity chromatography on a resin containing chelated Ni2+ after addition of an amino-terminal hexahistidine tag to the recombinant protein. Such purification is rapid and results in the isolation of highly purified protein in a single step. Furthermore, the introduction of a tobacco etch virus (TEV) polyprotein cleavage site between the hexahistidine tag and the G-protein α subunit permits the efficient removal of the tag by recombinant TEV protease.
|Original language||English (US)|
|Number of pages||19|
|Journal||Methods in Enzymology|
|State||Published - Jan 1 1994|
ASJC Scopus subject areas
- Molecular Biology