Recombinant expression and functional characterization of defensin-like peptide TEWP and its analogs in Pichia pastoris

Abstract

Defensin-like turtle egg white protein (TEWP) is a member of the defensin family identified in the vertebrate sea turtle (Caretta caretta). To increase the biological activity and reduce the toxicity of TEWP, we designed three analog peptides using different strategies, including amino acid residue substitution (MTEWP), peptide sequence reversion (RMTEWP), and conjugation with the cell-penetrating peptide ((RW)₄RMTEWP). The DNA fragments encoding the parent peptide TEWP and three analogs were tagged with C-terminal 6xhistidine and transformed into P. pastoris GS115 cells. The recombinant peptides were expressed in engineered P. pastoris cells under methanol induction for 96 h. At the end of the induction, the antimicrobial activities of the recombinant peptides were analyzed against 19 microbial strains. TEWP and its analogs showed the highest inhibitory effect against the L. monocytogenes ATCC 7644 strain (2-4 µg/ml). Increasing the net charge of the parent peptide to + 12 did not remarkably affect the antimicrobial activity. However, reversion of the peptide sequence and conjugation with a CPP resulted in a 2-fold increase in antimicrobial activity and a significant decrease in hemolytic activity. All recombinant peptides showed good resistance to heat treatment and protease degradation and maintained stability at high salt concentrations. The P. pastoris expression system developed in this study provides a robust biotechnological platform for the industrial-scale production of TEWP analogs, enabling their integration into pharmaceutical formulations such as smart hydrogels and targeted nanocarriers to enhance therapeutic delivery.

Keywords: Cell-penetrating peptide (CPP); Defensin-like peptide; Peptide modification; Pichia pastoris; Recombinant antimicrobial peptide; Retro-analog.