(A) Coomassie gel of 10 AMPs (L to R): B1CTcu1, PEP3, CA(1-7)M(2-9), BP100, Magainin 2, Cecropin P1, Cecropin B, Bac7(1-35), Tachystatin A1, Opistoporin 1. (–) indicates null reaction lacking DNA template. A bracket frames the molecular weight range of AMPs, while bands above the bracket originate from CF machinery.

(B) Coomassie gel of three peptides (L to R), Magainin 2, Opistoporin 1, and Cecropin P1, that were designed to contain (from N to C terminus) a leader sequence (L), affinity tag (His x6 or FLAG), Factor Xa cleavage site, and AMP sequence, allowing purification as described in (C). Lanes 1–3, His-tagged; lanes 4–6, FLAG-tagged; lanes 7–9, non-tagged versions. Arrows indicate relevant bands in lanes 1–6. Below lanes 1–3 and lanes 4–6 are WBs of the triplets with His or FLAG probes, respectively.

(C) Left: peptide purification scheme. Right: Coomassie gel demonstrating FLAG-based purification and cleavage for Cecropin P1. M, molecular weight ladder; (–), null reaction; (+), non-tagged Cecropin P1 size control; I, FLAG-tagged Cecropin P1 size control (blue arrow); II, purified FLAG-tagged Cecropin P1 (orange arrow); III, Factor Xa cleavage and release of the purified AMP (red arrow).

(D) Left: molecular weight filtration scheme of non-tagged AMPs (red circles). Transcription and translation machinery (black icon mix) retained on 10 kDa membrane, while non-tagged AMPs (red circle) pass through. Right: Coomassie gel of three example AMPs (L to R): Magainin 2, Cecropin P1, and Cecropin B.

(E) Chart showing growth inhibition assay of E. coli subjected to three individual AMPs (indicated in the chart legend) alongside untreated and null samples. OD 600 was measured every 30 min for 18 hr.

(F) Chart showing growth inhibition assay of B. subtilis subjected to one AMP, as described above.

All data points in (E) and (F) represent the mean ± SD of three biological replicates.