PURIFICATION OF ELONGATION-FACTORS EF-TU AND EF-G FROM ESCHERICHIA-COLI BY COVALENT CHROMATOGRAPHY ON THIOL-SEPHAROSE

The elongation factors EF-Tu and EF-G of Escherichia coli are involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively. Both possess cysteine residues th at are important for activity. We took advantage of this property to d esign 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 affini ty resin. Bacterial cells were lysed by a lysozyme-EDTA method, and th e lysate supernatant was purified by chromatography on, first, DEAE-Se phacel and, then thiol-Sepharose. Both elongation factors were purifie d in a single procedure, since DEAE-Sephacel fractions containing both factors were loaded on the thiol-Sepharose column. Thiol-Sepharose ch romatography efficiently separates each elongation factor from all con taminating proteins, The purified elongation factors were characterize d by SDS-PAGE, protein sequencing, and biological activity. The specif ic reactivities of the elongation factors with thiol-Sepharose allow t heir efficient purification and suggest that they possess hitherto und iscovered properties connected with their reactive thiols, (C) 1998 Ac ademic Press.