Iranian Research Organization for Science and Technology (IROST) Microbiology, Metabolites and Biotechnology 2980-8855 7 2 2024 12 01 Enhanced Expression of an Acinetobacter baumannii specific recombinant endolysin in Escherichia coli 102 108 1509 10.22104/mmb.2025.7277.1159 EN Fatemeh Khosravi Node Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran 0000-0002-5200-886X Nahid Bakhtiari Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran Somaye Imanparast Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran Davood Zare Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran Journal Article 2024 12 21 Prophage endolysin PlyF307, a peptidoglycan-destroying enzyme previously identified through the screening of the Acinetobacter baumannii genome, has shown the ability to kill numerous clinical isolates of <em>A. baumannii</em> in its recombinant form. <em>A. baumannii</em> is an extremely antibiotic-resistant Gram-negative hospital pathogen that is distributed worldwide. In this study, we used <em>Escherichia coli</em> BL21(DE3) and BL21(DE3) pLysS as a recombinant protein expression host to produce His-tagged PlyF307. Expression was done in Luria-Bertani (LB), Terrific Broth (TB), and auto-inducing medium, and different concentrations of β- d-1-thiogalactopyranoside (IPTG) were used for inducing. Induction was performed several times during the logarithmic growth phase. Bacterial cells were harvested at different post-induction times. Extraction and purification of the recombinant endolysin were performed using different lysis buffers and sonication programs. According to the experimental results, expression inducing was done with 0.1 mM IPTG at OD₆₀₀ = 0.9. The incubation temperature was 37 °C before and after the induction time. Finally, 520-570 mg of recombinant his-tagged PlyF307 (19.7 kD) was purified in different batches using 250 mM imidazole from 8- h post-induction harvested <em>E.coli</em> BL21(DE3) pLysS- PlyF307 cultured in 1- l Luria- Bertani broth (LB) medium in baffled flasks. The purified recombinant protein was verified using the western blotting technique. In conclusion, the strong positive net charge and bacteriolytic activity of the PlyF307 make it a suitable candidate for use in therapeutics and other biotechnology applications. Enhancement of the recombinant endolyzin production yield was considerable in this study and will be helpful to achieve this purpose, and this improved expression can be a significant step toward the scaling-up of the enzyme production in <em>E. coli</em>. https://mmb.irost.ir/article_1509_1cd3882394520876dc88d1472aa2a93f.pdf