| Summary: | Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (E<sub>W</sub>) from phage ΦX174 and the screened mutant protein E (E<sub>M</sub>) in the <i>Escherichia coli</i> BL21(DE3) strain. The results show that the lysis efficiency mediated by protein E<sub>M</sub> was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD<sub>600</sub> = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein E<sub>M</sub> was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in <i>Salmonella enterica</i> (<i>S. Enterica</i>, SE). A pET vector containing E<sub>M</sub> and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.
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