Secretory expression of mature recombinant elafin in Pichia pastoris and assessment of its in vitro antibacterial and antifungal effects

Elafin is a small human host-defense peptide derived from trappin-2/pre-elafin, known for its antiprotease and anti-inflammatory functions. However, direct comparative evidence on the antimicrobial activity of mature recombinant elafin, particularly against fungal organisms, remains limited. This study aimed to produce secreted mature recombinant elafin in Pichia pastoris X-33 and comparatively assess its antimicrobial activity in vitro against Escherichia coli ATCC 25,922, Pseudomonas aeruginosa ATCC 15,442, Staphylococcus aureus ATCC 29,737, and Candida albicans ATCC 10,231 using agar well diffusion, viable-count assays, broth microdilution, and minimum bactericidal/minimum fungicidal concentration (MBC/MFC) determination. The elafin insert was cloned into pPICZαA, and positive P. pastoris transformants were confirmed by colony PCR. Elafin was secreted into the culture medium, and the 66 h post-induction supernatant was selected for purification based on SDS-PAGE detection of a low-molecular-weight band in the expected size range. Purification resulted in a low-molecular-weight His-tagged recombinant product with migration consistent with the expected size of the designed elafin construct. In agar well diffusion assays, recombinant elafin produced inhibition zones of 32, 28, 25, and 22 mm for E. coli , P. aeruginosa , C. albicans , and S. aureus , respectively. Viable-count assays showed concentration- and time-dependent but modest reductions in recoverable cells, with all reductions remaining below 1 log₁₀ and the greatest effect observed at 62 µg/mL after 6 h. The minimum inhibitory concentration (MIC) values were 31 µg/mL for E. coli , P. aeruginosa , and C. albicans , and 62 µg/mL for S. aureus . The MBC values were 62 µg/mL for E. coli and P. aeruginosa , 124 µg/mL for S. aureus , and the MFC for C. albicans was 62 µg/mL. This study demonstrates the secretory expression and purification of a low-molecular-weight His-tagged recombinant product consistent with the designed mature elafin construct in P. pastoris . The purified peptide exhibited measurable in vitro inhibitory activity under defined assay conditions, supporting further evaluation of recombinant elafin in broader microbial and physiological models.