Isolation and functional analysis of histidine‐tagged elongation factor Tu

Abstract

The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF‐Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF‐Tu. Here we describe a novel system for the purification of EF‐Tu mutant species, based on metal‐ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF‐Tu with an additional C‐terminal sequence of one serine and six histidine residues. A cell extract containing the His‐tagged EF‐Tu (EF‐TuHis) is applied to a Ni2+ ‐nitrilotriacetic acid column. EF‐TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild‐type EF‐Tu. In‐vitro and in‐vivo functional analyses show that EF‐TuHis resembles the wild‐type EF‐Tu, which makes this one‐step isolation procedure a promising tool for the study of the interactions of mutant EF‐Tu with the various components of the elongation cycle. The new isolation procedure was succesfully applied for the purification of a mutant EF‐TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl‐tRNA. Copyright © 1992, Wiley Blackwell. All rights reserved