Amphibian-Derived Cathelicidin-DM and Cathelicidin-BG: Recombinant Overexpression in Escherichia coli and Comparison of Their Structures and Antimicrobial Activities.

Cathelicidin-DM and cathelicidin-BG are homologous and are expected to be similar in structure and biological activity. However, while all structural prediction tools tested showed cathelicidin-BG to be helical, there were mixed results for cathelicidin-DM, with most predicting it to contain three antiparallel β-sheets and no helices. Also, separate researchers, in nonidentical conditions, reported cathelicidin-BG to possess antimicrobial activity against only Gram-positive bacteria, unlike cathelicidin-DM, which affected both Gram-positive and Gram-negative bacteria, suggesting more dissimilarities between the peptides. We therefore decided to experimentally verify and compare the structures and activities of the peptides. Until now, there is no experimentally determined structural information on either peptide, and no cheap, efficient procedure has been reported for their mass production. We hereby report a recombinant method for cathelicidin-DM and cathelicidin-BG overexpression and purification, which yielded 1.19 and 1.92 mg of pure peptides, respectively, per 0.5 L of Luria-Bertani culture. At all oxidation states, both peptides adopted a random coil structure in sodium phosphate buffer (SPB) at pH 7.4, but switched to a helical conformation in membrane mimetics. In SPB, both native peptides demonstrated strong activity against both Gram-positive and Gram-negative bacteria, with ≤5 μM of each peptide killing all cells of the tested bacterial strains, through membrane disruption as one of the possible mechanisms. We therefore conclude that, under the conditions studied, both cathelicidins have comparable structures and antimicrobial activities as their sequence homology suggested; and we recommend the use of laboratory experimentations for validation of structural prediction results.