Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen

The structural gene for anthrax edema factor (EF) was expressed in Escheric hia coli under the control of a powerful T5 promoter to yield the 89-kDa re combinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate aff inity chromatography and cation-exchange chromatography. From 1 liter of cu lture, 2.5 mg of biologically active EF was easily purified. This is the fi rst report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for b inding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role o f these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138 , Ile140, and Lys142 of EF specifically blocked its interaction with anthra x protective antigen. The adenylate cyclase activity of the mutants remaine d unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, a nd Lys142 are required for binding of EF to anthrax protective antigen, whi ch facilitates its entry into susceptible cells.