Disulfide bond formation is not involved in cap-binding activity of Xenopus translation initiation factor eIF-4E

The eukaryotic initiation factor eIF-4E from Xenopus laevis was expressed in Escherichia coli and refolded in an active form. To define the cysteine residues forming a disulfide bond in Xenopus eIF-4E, each of the 3 cysteine residues was changed to serine by site-directed mutagenesis. Cap-binding activities of the mutant proteins were evaluated by 7-methyl-GTP(m7GTP)-affinity column chromatography. Even the mutant protein containing no cysteine showed an affinity for m7GTP. From the above results and the estimation of the sulfhydryl groups by Ellman's assay method, we concluded that a disulfide bond is not involved in the active Xenopus eIF-4E.