Enhanced Expression of an Acinetobacter baumannii specific recombinant endolysin in Escherichia coli

Document Type : Research Paper

Authors

Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran

Abstract

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 A. baumannii in its recombinant form. A. baumannii is an extremely antibiotic-resistant Gram-negative hospital pathogen that is distributed worldwide. In this study, we used Escherichia coli 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 E.coli 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 E. coli.

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References

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Microbiology, Metabolites and Biotechnology
Volume 7, Issue 2
December 2024
Pages 102-108

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