Purification of recombinant Rho/Rac/G25K from Escherichia coli

Abstract

This chapter discusses the purification method of recombinant Rho/Rac/G25K from Escherichia coli. The purification of Ras-related GTP-binding proteins from recombinant sources has proved to be invaluable for studying their biochemical properties and biological effects. The simplest expression systems have made use of Escherichia coli, although Ras-like GTPases produced in this way are not posttranslationally modified. Yeast and baculovirus-Sf9 (Spodoptera frugiperda, fall armyworm ovary) insect cells have also been used, and because they are eukaryotic hosts, the GTPases expressed are at least partially modified. The mammalian Rho subfamily consists of RhoA, B, and C; Rac1l and 2; G25K/CDC42; RhoG; and TC10. These proteins are 30% identical to Ras in amino acid sequence and 55% identical to each other, and their overall three-dimensional structure is similar to that of Ras. Like all small GTPases, the Rho-related proteins are regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and the characterization of these regulatory proteins relies on a source of a recombinant protein. © 1995, Elsevier Inc. All rights reserved.