Lethal factor (LF), a virulence factor of Bacillus anthracis, plays key roles in anthrax pathogenesis and host-pathogen interactions. The detailed mechanisms by which LF contributes to infection are still under investigation. While these studies require pure, homogeneous and reliable LF preparations, most methods reported for production of recombinant LF (rLF) in B. anthracis or Escherichia coli either are complicated or add extra residues to the protein. In this work, we modified our previous method by codon optimization and chromatograph workflow refinement and developed an improved strategy for efficient production of rLF from the periplasm of E. coli. We were able to obtain fully functional rLF with a purity above 95% and with a considerable yield of 5 mg/L. The preparation was characterized by SDS-PAGE, Western blot, and N-terminal sequencing, and the activity was validated by intoxication of macrophages and Fischer 344 rats. Our final product is suitable for most research involving drug development and mechanism analysis of anthrax pathogenesis. (C) 2013 Elsevier Inc. All rights reserved.