Purification of Elongation Factors EF-Tu and EF-G fromEscherichia coliby Covalent Chromatography on Thiol–Sepharose

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The elongation factors EF-Tu and EF-G ofEscherichia coliare involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively. Both possess cysteine residues that are important for activity. We took advantage of this property to design a purification protocol based on thiol–Sepharose chromatography, a method involving thiol-disulfide interchange between protein thiol groups and the glutathione-2-pyridyl-disulfide conjugate of the affinity resin. Bacterial cells were lysed by a lysozyme–EDTA method, and the lysate supernatant was purified by chromatography on, first, DEAE–Sephacel and, then thiol–Sepharose. Both elongation factors were purified in a single procedure, since DEAE–Sephacel fractions containing both factors were loaded on the thiol–Sepharose column. Thiol–Sepharose chromatography efficiently separates each elongation factor from all contaminating proteins. The purified elongation factors were characterized by SDS–PAGE, protein sequencing, and biological activity. The specific reactivities of the elongation factors with thiol–Sepharose allow their efficient purification and suggest that they possess hitherto undiscovered properties connected with their reactive thiols.