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

Articles in Press

Document Type : Research Paper

Authors

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

2 Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares sn, 33300 Villaviciosa, Asturias, Spain

Abstract

Prophage endolysin PlyF307, a peptidoglycan destroying enzyme previously identified through the screening of 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 distributed worldwide. In this study, we used Escherichia coli BL21(DE3) and BL21(DE3) pLysS, as recombinant protein expression host for producing of 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 at several times of growth logarithmic- phase. Bacterial cells were harvested in 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. Incubation temperature was 37 °C before and after 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 western blotting technique. In conclusion, the strong positive net charge and bacteriolytic activity of the PlyF307 makes 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 absolutely 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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Main Subjects

Microbiology, Metabolites and Biotechnology

Articles in Press, Accepted Manuscript
Available Online from 18 February 2025

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