Recombinant expression of antimicrobial peptide AMP-PD as tandem octamer and its antibacterial activity in vitro

The widespread misuse of antibiotics has led to increased bacterial resistance, posing a severe threat to public health. Antimicrobial peptides (AMPs) have emerged as promising alternatives due to their low resistance and broad-spectrum antibacterial activity. However, the low heterologous expression efficiency of AMPs has hindered their large-scale application. Herein, a novel tandemly gene duplication expression system of AMP was developed with recombinant plasmid His₆-AMP-PD₈ that containing eight OM19R repeats and tandemly linked by aspartic acid (D) and proline (P), using AMP-PD (PVDKPPYLPRPRPIRRPGGRD) as a model. Soluble expression of the fusion protein was successfully achieved in E. coli BL21(DE3). The expression efficiency of the fusion protein, including induction temperature, induction time, and IPTG concentration, was optimized to enhance the yield of the recombinant protein. About 32 mg/L the recombinant protein was achieved under the optimal condition, 35 °C, 0.7 mmol/L IPTG and 10 h of induction time. Then, 16 mg/L AMP-PD was obtained after cleavage with 50 % formic acid at 55 °C for 24 h and isolated using 3 kDa ultrafiltration device. Finally, the AMP-PD exhibited considerable antibacterial activity with 12.59 mm of inhibition circle diameter, and the MIC and MBC for E. coli ATCC25922 were 45.75 μg/mL and 62.53 μg/mL, respectively. This study successfully realized the soluble expression system of AMP-PD, laying the foundation for its industrial production and general application for AMP biosynthesis.