Engineering a Bacterial Platform for Efficient Expression of Histatin 5: A Promising Candidate for Antimicrobial Therapy

Introduction: Antibiotic resistance remains a key medical challenge, highlighting the need for alternative antimicrobial agents. Antimicrobial peptides are promising candidates due to their broad activity and high selectivity. Histatins produced by salivary gland cells are notable for their fungicidal, bacteriostatic, wound-healing, and immunomodulatory properties. Recombinant production of histatins may offer a more feasible approach than chemical synthesis. This study aimed to develop a bacterial expression system for histatin 5 (Hst5), including cassette constructs designed to enhance expression.

Methods: Plasmids pH5 and p10xH5 were constructed using the pET30(a) vector. Cloning was performed in E. coli XL1-Blue, followed by plasmid purification and restriction analysis. Peptide expression was performed in E. coli BL21(DE3) with IPTG induction. Peptides were detected by PAGE and purified using SP-Sepharose ion-exchange chromatography. Cassette peptide was hydrolyzed with cyanogen bromide to release Hst5 monomers. Peptide concentration was determined by the Bradford assay. Antimicrobial activity was assessed against E. coli using agar diffusion assay.

Results: Both vectors encoding the monomeric and cassette Hst5 were obtained. Optimal induction conditions were established (200 μM IPTG, 90 min), and Hst5 was eluted at 170–290 mM NaCl. Hydrolysis of the cassette form resulted in Hst5 monomers (280.7 μg/mL). The recombinant peptide displayed pronounced antimicrobial activity comparable to ampicillin.

Conclusions: The results demonstrate the feasibility of producing biologically active Hst5 in a bacterial expression system. This approach offers a cost-effective strategy for generating histatins, supporting the development of novel therapeutics against resistant pathogens. This work was supported by the Ministry of Science and Higher Education of the Russian Federation in 2025 (Agreement No. 075-15-2025-464).