Construction and application of plasmid-based opportunistic autolytic bacteria based on gene lys of MS2 bacteriophage

Abstract:

[Background] Conventional prokaryotic expression of heterologous protein usually requires ultrasonication or enzymolysis to lyse bacteria, the process of which is quite cumbersome. [Objective] We aimed to construct a type of plasmid-based opportunistic autolytic bacteria that exploited the lysis (lys) gene-mediated autolytic property of MS2 bacteriophage to facilitate the process of obtaining heterologous proteins. [Methods] The gene lys was cloned from the MS2 bacteriophage into a recombinant expression plasmid, which was heterologously-expressed in Escherichia coli BL21(DE3), and a novel type of plasmid-based opportunistic autolytic bacteria was generated. By L-arabinose induction, the lytic efficiency of the above-mentioned bacteria was assessed by growth curves and colony forming units (CFUs). Finally, the release levels of the heterologous protein were examined by SDS-PAGE. [Results] Three strains of plasmid-based opportunistic autolytic bacteria, pBAD- lys BL21(DE3), pBAD-Opti- lys BL21(DE3), and pCDF-BAD-Opti- lys BL21(DE3), were constructed. Following L-arabinose induction, all three strains of plasmid-based opportunistic autolytic bacteria achieved over 99.99% lytic efficiency. CFU data indicated that the host carrying pCDF-BAD-Opti- lys plasmid had better lytic effect than the other two. Upon L-arabinose-induced lysis of this host strain with heterologous expression of His-tagged enhanced green fluorescent protein (eGFP), more than 63% of eGFP were released to the outside of the cell and a single target protein of approximately 30 kDa was obtained directly from the culture medium by Ni-NTA. [Conclusion] In this study, we have successfully constructed a type of lys -mediated, plasmid-based opportunistic autolytic bacteria, which is capable of releasing most of the cytosolic heterologous proteins through L-arabinose-induced autolysis, simplifying the traditional process to acquire heterologous proteins.

Key words:MS2 bacteriophage;lys;autolytic bacteria;protein purification