Purification and Characterization of Colicin E1

Abstract

Abstract Colicin E1 was purified from mitomycin C-induced Escherichia coli JC411 (ColE1) by salt extraction of extracellular, bound colicin followed by ammonium sulfate fractionation and ion exchange chromatography. The purified preparation was considered homogeneous by several criteria. Gel filtration on Sephadex G-200 revealed no additional protein components and only a single band was observed upon cellulose acetate and isoelectric gradient electrophoresis. Sedimentation velocity and equilibrium studies, respectively, indicated the presence of a single molecular species with an s020,ω equal to 3.0 S and a molecular weight of 56,000. Double diffusion experiments using antisera prepared against purified colicin E1 yielded a single precipitin line in agar gel. Colicin E1 is a simple protein and no other moieties could be detected in association with the molecule. The isoelectric point (pI) of colicin E1, as determined by electrofocusing, is 9.05, and amino acid analysis revealed a high lysine and arginine content. A single NH2-terminal amino acid, methionine, was recovered in approximately equimolar yield per mole of protein. Circular dichroism spectra of purified colicins E1, E2, and E3 indicated a greater α-helix content in colicin E1 when compared to the other two colicins. Finally, high concentrations of purified colicin E1 were active against sensitive, immune, and resistant strains; however, relatively lower concentrations exhibited a normal activity spectrum.